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934 | https://bio-protocol.org/en/bpdetail?id=934&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Surface Polysaccharide Extraction and Quantification
CB Cedric Arthur Brimacombe
John Thomas Beatty
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.934 Views: 18628
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Microbiology Feb 2013
Abstract
Gram-negative bacterial cells possess two membranes - the inner cytoplasmic membrane and the outer membrane. The two membranes are distinct in their composition; the inner membrane is composed of a phospholipid bilayer, whereas the outer membrane (OM) is composed of an asymmetrical bilayer, with the outer leaflet containing lipopolysaccharide (LPS) (Raetz and Whitfield, 2002). Surface polysaccharides, such as LPS O-antigen, or capsular polysaccharide, are often tightly associated with the OM (Whitfield, 2006). This tight association can be used to generate a rough quantification of surface polysaccharides of Gram-negative bacterial cells, as the OM can easily be dissociated from cells without associated cell lysis (Brimacombe et al., 2013). The following method describes how to quickly extract and quantify OM-associated polysaccharides.
Keywords: Polysaccharide Capsule Quantification Outer-membrane
Materials and Reagents
Culture of bacterial cells (This procedure works only for Gram-negative bacteria, for example Escherichia coli, Pseudomonas aeurginosa, or Rhodobacter capsulatus. The outer membrane, specifically LPS, is essential for this procedure to work)
50 mM sodium chloride (NaCl) dissolved in deionized H2O
50 mM ethylenediaminetetraacetic acid (EDTA) (EMD Millipore, catalog number: 324503 )
Phenol (Fisher Scientific, catalog number: A92-100 )
93% sulfuric acid (Avantor Performance Materials, catalog number: 2900-10 )
Carbohydrate stock solution (see Recipes)
Equipment
Microcentrifuge
Microfuge tubes (ESBE, catalog number: ESB-ES00507C )
Spectrophotometer
Cuvettes
Glass test tubes
Glass pipettes
Procedure
Extraction of surface polysaccharides from Gram-negative bacteria
Grow bacteria to desired growth phase (generally stationary phase) in desired growth medium.
Note: Growth medium may affect surface polysaccharide levels, so the same media should be used for all experiments if possible.
Measure OD650 of culture; dilute to < 1 OD if necessary to get an accurate measurement.
Normalize cultures to OD650 of 2.0 (or to maximum OD that bacterial culture will grow to if it is less than 2.0).
Harvest 1 ml of each normalized culture by centrifugation at 14,500 x g for 5 minutes in a microcentrifuge.
Carefully remove supernatant with a pipette, discard tip.
Wash cells by re-suspending in 1 ml of 50 mM NaCl, pellet by centrifugation at 14, 500 x g for 5 minutes, remove supernatant.
Repeat step A6 four additional times (5 total washes).
Re-suspend cells in 1 ml of 50 mM EDTA, and incubate at 37 °C for 60 minutes (EDTA causes LPS to dissociate, thus releasing the OM from cells).
Pellet cells by centrifugation at 14,500 x g for 5 minutes, carefully remove supernatant and transfer to fresh microfuge tube (supernatant contains all surface polysaccharides, including LPS, capsule etc.).
Quantification of surface polysaccharides
Prepare carbohydrate standards by diluting carbohydrate stock solution into 1 ml aliquots of: 0, 30, 60, 90, and 120 μg/ml of carbohydrate (e.g. 970 μl of dH2O + 30 μl of 1 mg/ml stock solution to generate a 30 μg/ml standard).
Prepare clean, acid washed glass test tubes (for a suggested protocol, see Reference 4). Pipette 200 μl of standards, a 200 μl control of 50 mM EDTA, and all test samples into separate tubes.
Move to fume hood.
Add 200 μl of 5% phenol to all tubes, mix well by shaking.
Add 1 ml of 93% sulfuric acid; mix well by swirling (use caution).
Allow colour to develop for 10 minutes at room temperature (reaction should turn yellow; intensity depends on carbohydrate concentration). Additional mixing by gentle swirling every 2-3 minutes may help reaction proceed faster.
Measure OD490 of all reactions in a spectrophotometer; concentration of carbohydrates can then be calculated from the standard curve.
Note: If necessary, dilute reactions in dH2O to get accurate spectrophotometer readings.
Representative data
Recipes
Carbohydrate stock solution
50:50 mixture of 0.5 mg/ml each of sucrose and fructose
Final concentration of 1 mg/ml carbohydrate (molecular biology grade recommended)
Acknowledgments
The development of this protocol was funded by a grant to J.T.B. from the Canadian Institutes of Health Research.
References
Brimacombe, C. A., Stevens, A., Jun, D., Mercer, R., Lang, A. S. and Beatty, J. T. (2013). Quorum-sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA). Mol Microbiol 87(4): 802-817.
http://openwetware.org/images/9/9e/GLASSWARE_CLEANING_PROCEDURES.pdf
Raetz, C. R. and Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635-700.
Whitfield, C. (2006). Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem 75: 39-68.
Article Information
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© 2013 The Authors; exclusive licensee Bio-protocol LLC.
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Category
Microbiology > Microbial biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Polysaccharide
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935 | https://bio-protocol.org/en/bpdetail?id=935&type=0 | # Bio-Protocol Content
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Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo
Hsien-Tsung Lai
Cheng-Ming Chiang
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.935 Views: 22736
Reviewed by: Lin FangFanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Cell Jan 2013
Abstract
Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1-158 amino acid residues, called Venus-N, and its C-terminal 159-239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is proportional to the strength of protein-protein interaction (Morell et al., 2008); and fourth, the BiFC signals are derived from intrinsic protein-protein interaction, rather than from extrinsic fluorophores that may not reflect true protein-protein interaction due to their nonspecific association with cellular macromolecules or subcellular compartments. However, some limitations of BiFC include slow maturation (T1/2 ~ 1 hour) of an eventually stable BiFC complex (Hu et al., 2002), making it unsuitable for real-time observation of transient interaction that disappears prior to BiFC detection, and enhanced BiFC background at high expression levels due to fusion-independent association between two non-fluorescent fragments association. BiFC signals generated by in vivo protein-protein interaction can be validated by amino acid mutation introduced at the protein-protein contact surfaces. This imaging technique has been widely used in different cell types and organisms (Kerppola, 2006).
Keywords: BiFC Venus BRD4 P53 HCT116
Figure 1. Protein fragments of Venus (super enhanced YFP) constructs. A. Venus protein (amino acids 1-239; accession number: CAO79509) was dissected into two fragments at residue 158 to generate Venus N-terminus (top) and Venus C-terminus (bottom). B. Schematic drawing of Venus-N-p53 and Venus-C-PDID fusion fragments. Venus-N-p53 and Venus-C-PDID contain Venus-N-terminus and Venus-C-terminus fused respectively to p53 (amino acids 1-393; Gully et al., 2012) and the phosphorylation-dependent interaction domain (PDID, amino acids 287-530) of human Brd4 (Wu et al., 2013), in which a flexible linker containing two copies of Gly4Ser peptide is introduced to allow optimal space contacts between Venus-N-terminus and Venus-C-terminus and also to prevent steric hindrance between the Venus fragment and its fused protein of interest. AscI and XbaI indicate the positions of restriction enzyme-cutting sites used for generating fusions from PCR-amplified DNA fragments. An initiation codon for methionine (M) was added to allow translation of Venus-C-PDID. It should be noted that, although linker peptides ranging from 5 to 17 amino acids are often used (Remy and Michnick, 2007), the exact length and the sequence nature of the linkers have not been systematically analyzed (Kerppola, 2013). C. BiFC fluorescence signal is produced when Venus-N and Venus-C are in close proximity brought together via p53-PDID interaction in the cell.
Materials and Reagents
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
Antibiotics (Penicillin/Streptomycin) (Sigma-Aldrich, catalog number: P0781 )
Cell culture medium (Complete: With 10% FBS and antibiotics; Antibiotic-free: with 10% FBS only)
Formaldehyde (Thermo Fisher Scientific, catalog number: F79-500 )
Triton X-100 (Sigma-Aldrich, catalog number: 79284 )
BSA (Sigma-Aldrich, catalog number: A3059 )
Lipofectamine 2000 (Life Technologies, Invitrogen™, catalog number: 11668-019 )
Venus-N-p53 (Gully et al., 2012) (Santa Cruz, catalog number: sc8334 ) and Venus-C-PDID (Wu et al., 2013) plasmids (Santa Cruz, catalog number: sc5384 ) (see Figure 1B)
Primary antibodies against Venus
e.g. Anti-full-length-GFP antibody (Santa Cruz, catalog number: sc8334 or sc9996 )
Anti-C-terminal GFP antibody (Santa Cruz, catalog number: sc5384)
or β-actin (Sigma-Aldrich, catalog number: A5441 )
Secondary antibody conjugated with a fluorescence dye emitting wavelength other than that of Venus (excitation 488 nm, emission 515 ± 15 nm) or Hoechst 33258 (excitation 350 nm, emission 461 nm), for example, Alexa Fluor® from Life Technologies
Sodium chloride (Thermo Fisher Scientific, catalog number: 7647-14-5 )
Potassium chloride (Thermo Fisher Scientific, catalog number: 7447-40-7 )
Sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O) (Thermo Fisher Scientific, catalog number: S373500 )
Potassium phosphate monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: 7778-77-0 )
Aluminum foil (grocery store)
Permanent mounting medium (Vector Laboratories, catalog number: H-5000 )
Microscope slides (Thermo Fisher Scientific, catalog number: 12-544-7 )
Nail polish (grocery store)
10x Phosphate Buffered Saline (PBS) (see Recipes)
3.7% Formaldehyde (freshly prepared) (see Recipes)
Phosphate Buffered Saline with Triton X-100 and BSA (PBSTB) (see Recipes)
Hoechst 33258 (Sigma-Aldrich, catalog number: 861405 ) (see Recipes)
Equipment
Glass bottom culture dish (35-mm glass bottom plate containing a 14-mm center microwell, poly-D-lysine coated) (MatTek, catalog number: P35GC-1.5-14-C )
Tissue culture hood (NuAire, model: Class II, Type A2 )
37 °C cell culture incubator (Thermo Fisher Scientific, Forma®, model: Series II , water-jacketed and HEPA filtered)
Confocal fluorescence microscope (Nikon, model: Eclipse TE-2000E/C1 )
Rocker (Labnet International, model: Rocker 25 )
Software
NIS Elements Basic Research (version 2.2)
Nikon EZ-C1 Free Viewer (version 3.90)
Procedure
Note: Steps 1 to 7 performed in a tissue culture hood; steps 8 to 10 done on regular bench.
The day before transfection: Seed log-phase growing cells of interest (2 x 105 cells in 2 ml) in a 35-mm glass bottom culture dish and allow overnight incubation for proper cell attachment and expansion in a 37 °C cell culture incubator.
Note: Optimum cell cultures are 30% to 40% confluent with a low percentage of overlapping cells on the day of transfection.
Note: Pre-warm culture medium and 1x PBS to 37 °C.
Rinse cells twice with 2 ml of 37 °C 1x PBS.
Note: Avoid center glass area when pipetting solutions at all steps.
Replace with 1 ml antibiotic-free medium.
Co-transfect Venus-N-p53 and Venus-C-PDID constructs with Lipofectamine 2000 according to manufacture's instructions.
Note: 0.5 μg of each construct (total 1 μg DNA) plus 2.5 μl of Lipofectamine 2000 in 25 μl of Opti-MEM works well with HCT116 cells.
Note: Negative controls, such as Venus-N-p53 with Venus-C linked to a non-interacting protein (or domain, e.g., Brd4 amino acids 149-284 described in Wu et al., 2013), Venus-N-p53 with Venus-C, Venus-N with Venus-C-PDID, or Venus-N with Venus-C, should be included in parallel for comparison.
Leave cells at 37 °C in a cell culture incubator.
Replace transfection medium with complete medium 6 hours post-transfection.
Incubate cells for 24 h at 37 °C in a cell culture incubator.
Note: Incubation time after transfection can vary by the level of protein expression. Pilot experiments to test the optimum expression time and the levels of protein expression for Venus-N-p53 and Venus-C-PDID are beneficial (see Figure 2).
Note: The amounts of transiently expressed proteins should be titrated to the levels of the endogenous proteins, reflecting endogenous protein interaction in vivo.
Figure 2. Venus-N-p53 and Venus-C-PDID protein expression. Western blot analysis of Venus protein expression in p53-null (p53-/-) HCT116 cells, 24 hours post-transfection. Antibodies: Venus-N-p53, Venus-C-PDID, and β-Actin.
Wash cells for 5 min with 2 ml of 1x PBS on a rocker at a speed of 10-20 rpm, total three times.
Note: This step is important to reduce the background signals of Hoechst 33258 due to non-specific adherence of transfection DNA deposits to the plate surfaces.
Prepare for cell imaging:
For live-cell imaging:
Note: Perform steps a to c (see below) on a rocker at the speed of 10-20 rpm.
Note: Avoid center glass area when pipetting solutions at all steps.
Stain DNA in the nucleus with Hoechst 33258, 2 ml (5 μg/ml), for 30 min at room temperature.
Note: For multiple dishes, prepare one dish at a time before next Hoechst 33258 staining so there is enough time for fluorescence microscope visualization.
Remove staining solution and wash cells for 5 min with 2 ml of 1x PBS, total three times.
Add 1 ml of 1x PBS for fluorescence detection.
Visualize fluorescence signals under a fluorescence microscope.
Acquire lower magnification images and then higher magnification images of bright field, Venus, and Hoechst 33258.
Note: This step may take up to 30 to 60 min depending on adjusting position/focus and higher resolution of images desired.
Typical results are shown in Figure 3.
Figure 3. BiFC results. Direct visualization of p53-PDID interaction in vivo by BiFC live-cell imaging performed with p53-null (p53-/-) HCT116 cells transiently expressing Venus-N-p53 and Venus-C-PDID. A. Two different magnification images were obtained by using the 10x ocular lens in combination with a 20x or 60x objective lens. A 20x objective lens is typically used for observing a large number of cells and providing a general glimpse of cellular localization, whereas a 60x objective lens allows more detailed localization within subcellular compartments (Kerppola, 2006). Merge: combined Venus (pseudo-colored green), Hoechst 33258 (pseudo-colored blue), and bright field signals. B. Magnified images of p53-PDID interaction shown in A (i and ii) from 600x images. The BiFC signals co-localize with nuclear DNA staining (Hoechst 33258) as presented in pseudo-colored cyan. Images were obtained by Nikon Eclipse TE-2000E/C1 confocal fluorescence microscopy using NIS Element Basic Research software and further processed by Nikon EZ-C1 software.
Note: Fixed cell images are virtually the same as live-cell imaging (data not shown).
For fixed-cell imaging:
Note: Perform steps a to k (see below) on a rocker at a speed of 10-20 rpm.
Note: Avoid center glass area when pipetting solutions at all steps.
Fix cell with 1 ml 3.7% formaldehyde in PBS for 15 min at room temperature.
Note: 3.7% formaldehyde must be freshly prepared.
Remove formaldehyde solution and wash fixed cells 5 min with 2 ml of 1x PBS, total three times.
Remove wash solution and incubate fixed cells with 2 ml of 1x PBS containing 0.25% Triton X-100 to permeabilize cells for 30 min at room temperature.
Wash cells for 5 min with 2 ml of 1x PBS, total three times.
Note: Skip antibody incubation procedures (steps e to i) if only signals from Venus and Hoechst 33258 (i.e., without fluorophore-conjugated antibody amplification) are needed. However these steps are helpful for verifying protein expression in transfected cells.
Incubate permeabilized cells with 2 ml of PBSTB for 30 min to block non-specific antibody binding.
Replace PBSTB with primary antibody (against protein of interest or GFP) in 1 ml of PBSTB for 1 h at room temperature or overnight at 4 °C.
Note: Start with 1:500 antibody dilution to determine the best condition. Two primary antibodies can be used at the same time.
Wash cells for 5 min with 2 ml of 1x PBS, total three times.
Incubate with secondary antibody (conjugated with fluorophores) in 1 ml of PBSTB for 1 hour at room temperature in the dark (wrap with aluminum foil).
Note: Start with 1:1,000 antibody dilution to determine best condition.
Wash cells for 5 min with 2 ml of 1x PBS, total three times.
Stain cell nucleus DNA with Hoechst 33258, 2 ml (0.5 μg/ml), for 10 min.
Wash cells for 5 min with 2 ml of 1x PBS, total three times.
Add 1 ml of 1x PBS for fluorescence detection.
Visualize reconstituted fluorescence signal under a fluorescence microscope.
Acquire lower magnification images and then higher magnification images of bright field, Venus, Hoechst 33258, or other signals from fluorophore-conjugated secondary antibody.
When needed, permanent preservation of samples can be done by the following steps:
Remove PBS and use razor blade to separate the bottom glass (i.e., coverslip) from the petri dish (see Figure 4A).
Note: Do not touch the cell-attached side in the center circle of coverslip.
Drop 50 μl of mounting medium on a microscope slide (see Figure 4B).
Gently tilt the coverslip (see Figure 4C), with the cell-attached side facing down, to mount with mounting medium on a microscope slide (see Figure 4D).
Gently press coverslip in the center with a pipet tip to remove air bubbles (see Figure 4E) and remove excess mounting medium around the edges with a paper towel (see Figure 4F).
Mark microscope slides and seal coverslip with nail polish around the edges for 15 min, or until dry, to prevent sample movement and drying (see Figure 4G).
Store at -20 °C or 4 °C in the dark.
Figure 4. Illustration of permanent preservation of BiFC samples
Recipes
10x PBS (1 L)
80 g NaCl
2 g KCl
21.7 g Na2HPO4.7H2O
2 g KH2PO4
Add ddH2O to 1 L
Autoclave and stored at room temperature
Dilute with ddH2O to make 1x PBS (autoclave required, then store at room temperature)
3.7% Formaldehyde (freshly prepared for 10 ml)
1 ml 37% Formaldehyde
1 ml 10x PBS
8 ml sterilized ddH2O
PBSTB (freshly prepared for 100 ml)
10 ml 10x PBS
250 μl Triton X-100
1 g BSA
Add sterilized ddH2O to 100 ml
Hoechst 33258
Dissolve in 1x PBS to make final stock concentration 10 mg/ml
Dilute to desired concentration with 1x PBS
Stored at 4 °C
Acknowledgments
We thank Dr. Shwu-Yuan Wu for technical help and discussions during the development and writing of this protocol. The protocol detailed here was extended primarily from the procedures described in Wu et al. (2013). This work was supported in part by NIH grants (CA103867 and CA124760), CPRIT grants (RP110471 and RP120340), and a Welch Foundation grant (I-1805).
References
Ding, Z., Liang, J., Lu, Y., Yu, Q., Songyang, Z., Lin, S. Y. and Mills, G. B. (2006). A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc Natl Acad Sci U S A 103(41): 15014-15019.
Gully, C. P., Velazquez-Torres, G., Shin, J. H., Fuentes-Mattei, E., Wang, E., Carlock, C., Chen, J., Rothenberg, D., Adams, H. P., Choi, H. H., Guma, S., Phan, L., Chou, P. C., Su, C. H., Zhang, F., Chen, J. S., Yang, T. Y., Yeung, S. C. and Lee, M. H. (2012). Aurora B kinase phosphorylates and instigates degradation of p53. Proc Natl Acad Sci U S A 109(24): E1513-1522.
Hu, C. D., Chinenov, Y. and Kerppola, T. K. (2002). Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9(4): 789-798.
Kerppola, T.K. (2006). Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protocols 1(3): 1278-1286.
Kerppola, T. K. (2013). Design of fusion proteins for bimolecular fluorescence complementation (BiFC). Cold Spring Harb Protoc 2013(8): 714-718.
Morell, M., Espargaro, A., Aviles, F.X., and Ventura, S. (2008). Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry. Nat Protocols 3(1): 22-33.
Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K. and Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1): 87-90.
Remy, I. and Michnick, S.W. (2007). Application of protein-fragment complementation assays in cell biology. BioTechniques 42(2): 137-145.
Wu, S. Y., Lee, A. Y., Lai, H. T., Zhang, H. and Chiang, C. M. (2013). Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting. Mol Cell 49(5): 843-857.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Lai, H. and Chiang, C. M. (2013). Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo. Bio-protocol 3(20): e935. DOI: 10.21769/BioProtoc.935.
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Category
Cell Biology > Cell imaging > Fluorescence
Molecular Biology > Protein > Protein-protein interaction
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Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions
Marie Boutet
KF Katarzyna Franciszkiewicz
Audrey Le Floc’h
Fathia Mami-Chouaib
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.936 Views: 9661
Reviewed by: Lin FangFanglian He Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in Cancer Research Jan 2013
Abstract
This method allows evaluating the relative adhesion strength between T lymphocytes and specific adherent target cells using a shear force in flow chambers. It is based on the measure of the resistance of conjugates formed between T cells and adherent tumor cells to shear stress in a microfluidic system. For this purpose, T cells, stained with a CellTracker probe, are added into flow channels containing a monolayer of adherent target cells and their progressive detachment under a constant shear stress is then recorded using a fluorescent microscope.
Materials and Reagents
Adherent tumor cells [such as non-small cell lung carcinoma (NSCLC) cell lines]
Specific T-cell clones (generated either from autologous tumor-infiltrating T lymphocytes (TIL) or peripheral blood lymphocytes (PBL))
RPMI 1640 (Life Technologies, Gibco®, catalog number: 61870044 )
DMEM-F12 (Life Technologies, Gibco®, catalog number: 31331093 )
UltroserG (Pall, catalog number: 15950-017 )
Fetal Bovin Serum (Life Technologies, Gibco®, catalog number: 10270-106 )
Human serum AB (Institut de Biotechnologies Jacques Boy)
Penicillin and streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
Sodium Pyruvate (Life Technologies, Gibco®, catalog number 11360029 )
IL-2
10x PBS (Life Technologies, Gibco®, catalog number: 70011-036 )
CellTracker probe (CellTrackerTM Green CMFDA) (Life Technologies, Invitrogen™, catalog number: C2925 )
Complete DMEM: tumor cell culture medium (LC medium) (see Recipes)
RPMI-based T-cell complete medium (see Recipes)
Equipment
Microscope Zeiss LSM-510 (ZEISS) with a heated incubation chamber and CO2 supply
Micro-Slides VI, ibiTreat (ibidi GmbH, catalog number: 80606 ), two silicon tubes (1.6 mm of inner diameter) with a plastic clip, two Elbow Luer connectors (ibidi GmbH, catalog number: 80646 )
Syringe pump (high flow rate > 50 ml/min)
60 ml syringe (Becton, Dickinson and Company, catalog number: 300866 )
Humidified incubator at 37 °C with 5% CO2
A recipient for waste flow buffer (Erlenmeyer)
Centrifuge (Beckman Coulter, model: GS-6R )
Procedure
Adherent tumor cell preparation
Seed adherent tumor cells into IBIDI channels by adding 60 μl of tumor cell suspension in LC medium. Micro-slides VI, ibiTreat characteristics are the following:
Number of channels: 6
Minimal volume per channel: 30 μl
Channel length: 17 mm
Channel width: 3.8 mm
Channel height: 0.4 mm
Growth area: 0.6 cm2 per channel
Tumor cell concentration may vary according to the cell type (for instance: 1.6 x 106 cells/ml for NSCLC cell lines described in Reference 1). Cells should be at 90-95% of confluence the day of experiment.
Incubate the IBIDI slide in a humidified incubator at 37 °C for at least 2 h for cell attachment.
Fill gently the reservoirs with another 60 μl of LC medium. Avoid pipetting directly into the channels not to detach the cells.
Incubate overnight at 37 °C, 5% CO2.
Note: In case of tumor cell treatment (example siRNA transfection), cells should be plated two days before using the same experimental conditions. Medium may need to be changed every 24 h. Be sure that the cells are all alive and just reaching 90-95% confluence the day of experiment.
T-cell preparation
Wash T cells with PBS 1x by centrifugation at 350 x g for 5 min.
Stain cells with CellTracker Green (CMFDA) according to the manufacturer’s protocol. Briefly, resuspend cells at 2 x 106/ml in PBS and add one volume of CellTracker Green (CMFDA 2x) diluted in PBS (final concentration 1 μM). Incubate for 15 min at 37 °C.
Wash T cells twice with RPMI-based complete medium by centrifugation at 350 x g, 5 min.
Resuspend T cells in T-cell medium, at final concentration 2 x 106 cells/ml, in 24 flat bottom well plates.
Incubate T cells overnight in humidified incubator at 37 °C with 5% CO2.
T-cell adhesion strength under flow conditions
The following day, warm the thermostatic chamber of the microscope at 37 °C and 5% CO2 (Figure 1A).
Equilibrate RPMI-based complete medium (500 ml) inside the incubator at 37 °C and 5% CO2.
Prepare syringe pump (Figure 1B).
Connect the tube carrying a plastic clip (position closed) to the syringe and fill the syringe with prewarmed medium. Put the Elbow Luer connector and prime the tube (Figure 1A).
Put the IBIDI slide (Figure 1C) under the microscope objective (20x) (Figure 1D). Be sure that the reservoirs are completely full. If not, add some medium.
Connect the tube to one extremity of the IBIDI channel making sure there are no air bubbles remaining inside. This step is critical, because bubbles increase the risk of tumor cell detachment, influence the flow rate and can even stop the flow.
Figure 1. Flow system. A. Whole flow system; B. Pump system; C. Details of Micro-Slides IBIDI connections; D. During acquisition, the slide is fixed under the microscope and connected to the pump system.
Use the second tube to connect the opposite extremity of the channel with a bottle collecting wastes (Figure 2).
Red: adherent tumor cell layer
Green: T cells
Blue: LC medium
White arrow: direction of the flow
Figure 2. T-cell adhesion under flow conditions. Stained T lymphocytes were incubated for 15 min on a monolayer of autologous tumor cells previously seeded into IBIDI channels. The IBIDI slide is then connected by silicon tubes, in one side to a pump (with a syringe filled with prewarmed medium) and in the other side to the waste recipient.
Release the clip on the tube connecting the syringe to the IBIDI slide (Figure 1A).
Note: Test the system to validate the maximal flow rate that doesn’t detach tumor cells. This rate will be applied to determine the T-cell adhesion strength on tumor cells.
Wash T cells with RPMI by centrifugation at 350 x g for 5 min.
Resuspend T cells in RPMI medium at final concentration 2 x 106 cells/ml.
Replace the medium filling the channels with 50 μl of T cells suspension. Be careful to not detach tumor cells or introduce bubbles inside channels.
Incubate 15 min at 37 °C.
After incubation, prepare the flow system using the same conditions described for the test assay.
Add 50 ml pre-warmed medium inside the syringe.
Start the acquisition just before the flow (Figure 1D).
Acquire images every 2 s for 60 s. It is expected that T cells adhere more firmly to tumor cells that express adherence molecules (such as ligands for integrins expressed by T cells) than tumor cells that do not express these molecules. (Figure 3)
Figure 3. Representative images acquired at different time lapses during T cell adhesion protocol. A. 0 sec; B. 250 sec; C. 640 sec at the flow rate of 100 ml/h.
Recipes
Complete DMEM: tumor cell culture medium (LC)
DMEM-F12 supplemented with:
10% decomplemented Fetal Bovine Serum
1% UltroserG
1% Penicillin and streptomycin
1% Sodium pyruvate
RPMI-based T-cell complete medium
RPMI 1640 complemented with 10% Human serum AB
1% Penicillin and streptomycin
1% Sodium pyruvate
IL-2 (100 U/ml)
Acknowledgments
We thank Sophie Salomé-Desmoulez for her help with confocal microscopy. This work was supported by grants from the INSERM, the Association pour la Recherche sur le Cancer (ARC), the Institut National du Cancer (INCa), the Ligue contre le Cancer and the Cancéropôle Ile de France (IDF). MB is a recipient of a fellowship from the Cancéropôle IDF.
References
Bernard, G., Raimondi, V., Alberti, I., Pourtein, M., Widjenes, J., Ticchioni, M. and Bernard, A. (2000). CD99 (E2) up-regulates α4β1-dependent T cell adhesion to inflamed vascular endothelium under flow conditions. Eur J Immunol 30(10): 3061-3065.
Franciszkiewicz, K., Le Floc'h, A., Boutet, M., Vergnon, I., Schmitt, A. and Mami-Chouaib, F. (2013). CD103 or LFA-1 engagement at the immune synapse between cytotoxic T cells and tumor cells promotes maturation and regulates T-cell effector functions. Cancer Res 73(2): 617-628.
Rosenthal-Allieri, M. A., Ticchioni, M., Breittmayer, J. P., Shimizu, Y. and Bernard, A. (2005). Influence of β1 integrin intracytoplasmic domains in the regulation of VLA-4-mediated adhesion of human T cells to VCAM-1 under flow conditions. J Immunol 175(2): 1214-1223.
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Measurement of Junctional Protein Dynamics Using Fluorescence Recovery After Photobleaching (FRAP)
Rashmi Priya
Guillermo A. Gomez
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.937 Views: 13225
Reviewed by: Lin FangFanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Cell Biology Aug 2012
Abstract
Fluorescence Recovery After Photobleaching (FRAP) (Lippincott-Schwartz et al., 2003; Reits and Neefjes, 2001) was employed to determine dynamic properties of proteins localized at the ephitelial zonula adherens (ZA) (Kovacs et al., 2011; Otani et al., 2006). The proteins of interest were expressed in cells using a knockdown and reconstitution approach in which endogenous proteins were depleted by RNA interference (RNAi) and replaced by expression of an RNAi-resistant gene fused to GFP (Priya et al., 2013; Smutny et al., 2010; Smutny et al., 2011; Vitriol et al., 2007). By choosing expression levels of GFP-tagged proteins that were comparable to endogenous levels, we minimized transient overexpression artifacts due to overcoming regulatory mechanisms that directly affect protein dynamics (Goodson et al., 2010). Using this approach, junctional E-cadherin-GFP or GFP-Ect2 were subjected to FRAP analysis in small areas corresponding to the ZA using confocal microscopy (Priya et al., 2013; Ratheesh et al., 2012; Gomez et al., 2005; Trenchi et al., 2009). Although in principle this approach is similar in every case, bleaching conditions, acquisition parameters and analysis details might differ depending on the time scale of the recovery process (Lippincott-Schwartz et al., 2003). In this protocol we will describe the experimental procedure to perform FRAP experiments and how to optimize bleaching and acquisition conditions for optimal measurements of protein dynamics at cell-cell junctions.
Keywords: E-cadherin FRAP Cell-cell junctions Turnover Half life
Materials and Reagents
MCF-7 cells, mammary carcinoma epithelial cells derived from metastatic site (ATCC® HTB22 TM)
HEK293T cells
Plasmids
pLL5.0 lentiviral vector (Figure 1) and packaging plasmids pMDLg/pRRE, pMD2.G (VSV G) and pRSV-Rev. pLL5.0 is a modified version of pLL3.7 and it was generously provided by Jim Bear, Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599 (Vitriol et al., 2007; Rubinson et al., 2003)
pLL5.0 containing both a shRNA against the ORF of human CDH1 (NM_004360) (5′-GGGTTAAGCACAACAGCAA-3′) cloned downstream of the U6 promoter (HpaI and XhoI) (Figure 1) and a mouse E-Cadherin(NM_009864)-GFP fusion construct cloned at SacII and SbfI sites. The E-cadherin-EGFP fusion protein expression was driven by a 5’LTR promoter to facilitate lower expression levels of GFP fusion proteins for imaging (Smutny et al., 2011 )
pLL5.0 containing both a shRNA against the 3’UTR of human ECT2 (NM_001258315) (5'-GCTGTTTCAAAGTGTGATA-3') and cloned downstream of the U6 promoter (HpaI and XhoI) (Figure 1) in a modified version of pLL5.0. In this modified pLL5.0 the GFP reporter was replaced by the sequence that encompasses both the coding region for GFP and the multiple cloning site of pEGFP-C1 (Clontech) using EcoRI and SbfI sites. These restriction sites were not preserved after this cloning step. Then the human ECT2 coding sequence (NM_001258315) was cloned into the vector using EcoR1 and BamH1 sites (pLL5.0 GFP–shRNA resistant ECT2)
Figure 1. Schematic of pLL5.0 vector. Sites HpaI and XhoI are used for the subcloning of shRNA sequences desired to knockdown endogenous levels of the protein of interest. The U6 promoter drives the expression of this shRNA sequence. Contrarily, a shRNA resistant version of the same protein can be subcloned downstream of the 5’LTR promoter and fused to GFP. Thus, it is possible to achieve endogenous levels of expression for a fluorescent-tagged protein and preventing effects associated to its overexpression. MCS = Multiple cloning site
Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Gibco, catalog number: 11995-073 )
Foetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 26140079 )
Phosphate buffered saline (PBS) without Ca2+ and Mg2+ (Astral Scientific, catalog number: 09-8912-100 )
16% Paraformaldehyde (formaldehyde) (PFA) aqueous solution (ProSciTech, catalog number: C004 )
Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
In-Fusion cloning kit (Clontech, catalog number: 638909 )
Hank’s Balanced Salt Solution (Sigma-Aldrich, catalogue number: H8264 )
2.5% Trypsin (10x) (Life Technologies, catalogue number: 15090046 )
Note: This solution is diluted to 0.25% final concentration with PBS.
Poly(vinylidene difluoride) spin columns (Amicon Ultra Centrifugal filters, UltraCel-100K) (EMD Millipore, catalog number: UFC910024 )
Sodium butyrate (Sigma-Aldrich, catalogue number: B5887 ) (see Recipes)
Hexadimethine bromide (polybrene) (Sigma-Aldrich, catalog number: H9268 ) (see Recipes)
Imaging media (see Recipes)
4% Paraformaldheyde in PBS (see Recipes)
Equipment
25 cm2 Nunclon Delta Flasks (Thermo Fisher Scientific, Nunc®, catalog number: 156367 )
175 cm2 Nunclon Delta Flasks (Thermo Fisher Scientific, Nunc®, catalog number: 159910 )
Laser scanning confocal microscope equipped with acousto-optic tunable filters (AOTF) for bleaching of selected areas and heated chamber (37 °C) for live cell imaging. The microscope must also be equipped with dichroic and emission filter for the use of the 405 and 488 nm laser lines and detection of GFP fluorescence. The experiments shown were performed on LSM 510 Meta or LSM 710 inverted confocal microscopes (ZEISS)
30 mW Argon (458, 488 and 514 nm laser lines) and 25 mW (405 nm) diode lasers (LASOS Lasertechnik GmbH)
Plug-in FRAP profiler (McMaster University, Canada)
Glass bottom dishes (#1.5) (MatTek, catalog number: P35G-1.5-20-C or Shengyou Biotechnology, catalog number: D29-10-1.5-N)
Software
Image J software
Prism, GraphPad
Matlab, MathWorks
Procedure
Cell preparation
Expression of GFP-tagged proteins in a knockdown background
We have used this approach in our recent article published in Nature Cell Biology (Ratheesh et al., 2012) to characterize the dynamic properties of the adhesion molecule E-cadherin and the RhoA GEF, Ect2. For the expression of these proteins at endogenous levels, we used the pLL5.0 lentiviral vector (Vitriol et al., 2007; Rubinson et al., 2003). This vector contains two promoters, a U6 promoter that drives the expression of shRNA and a 5’LTR promoter that drives the expression of a shRNA-resistant gene (Figure 1).
Lentivirus preparation and viral transduction
HEK293T cells were cultured in 20 ml DMEM supplemented with 10% FBS at 37 °C and maintained under these condition during the following steps.
Constructs made in the pLL5.0 vector were simultaneously transfected with packaging vectors into HEK-293T cells by CaCl2 precipitation.
48 h after transfection, cells were treated with sodium butyrate (10 mM final concentration) to increase gene induction.
Virus-like particles (VLPs) were harvested 48–72 h after transfection and concentrated on poly(vinylidene difluoride) spin column as follows:
Collect media of cells and spin down in 50 ml conical tube.
Filter the supernatant into new tubes using 0.2 μm syringe filters.
Add 10 ml filtrate to the poly(vinylidene difluoride) spin column and centrifuge at 3,200 rpm on a bench top centrifuge for 20 min at room temperature. This will reduce the volume of the suspension of VLPs to ~800 μl per tube.
Discard the flow trough and add the remaining supernatant (~10 ml) to the the poly(vinylidene difluoride) spin column and repeat the above step.
Aliquots of virus were subsequently used for titration or stored at -80 °C. Titers were determined as described before (Smutny et al., 2010).
Preparation of the cells for image acquisition
For FRAP experiments, MCF-7 cells were cultured in DMEM supplemented with 10% FBS and infected with lentiviral particles at a multiplicity of infection of 10 per cell on 25 cm2 flasks.
Cells were incubated at 37 °C with the lentivirus in DMEM + FBS and Polybrene (8 μg ml-1) and harvested by trypsinization three days after infection.
Single-cell suspensions were seeded on glass bottom dishes at 80% confluence and allowed to grow for 48 h (or until they reach full confluence) for FRAP experiments.
Prior to image acquisition, cells were washed with imaging media and incubated with 1.5 ml of it for the duration of the experiment.
Image Acquisition
FRAP experiments were performed on a LSM 510 Meta or LSM 710 Zeiss confocal microscope for E-cadherin-GFP or GFP-Ect2, respectively. Microscopes were equipped with a heated stage maintained at 37 °C and a 30 mW Argon laser (458, 488 and 514 nm laser lines). The LSM 710 Zeiss confocal microscope was also equipped with a 405 nm (25 mW) diode laser. Images (pre and post-bleach, Figure 1) were acquired using 60x objective, 1.4 NA oil Plan Apochromat immersion lens at 4x digital magnification with 0.7 μm optical section. A 488 nm laser line of an argon laser (30 mW) was used for fluorescence excitation at 1-3% transmission.
For E-cadherin-GFP dynamics, time-lapse images (416 x 416 pixels, 0.086 μm/pixel) were acquired before and after photobleaching with an interval of 5 seconds per frame for the total time of 280 seconds (Figure 1A). A constant region of interest (ROI) of 2.8 x 1.7 μm with the longer axis parallel to the cell-cell contact was marked for each experiment and E-cadherin-GFP was bleached with 50 iterations of the 488 nm laser with 100% transmission. This resulted in maximum bleach of approximately 70%.
Ect2 dynamics was assessed using GFP-Ect2 co-expressed with Ect2 shRNA by lentiviral infection. A constant circular ROI (1.4 μm diameter) in approximately the center of the cell-cell contact was bleached to ~ 70% with both the 488 and the 405 nm lasers turned on simultaneously at 100% transmission. Time-lapse images of the same region were acquired before (20 frames, 5 seconds) and after (210 frames, 50 seconds) photobleaching with an interval of ~ 250 m per frame (Figure 1B).
For these experiments, cells with slanted contacts were chosen which allowed us to precisely identify and photobleach the ZA.
Special considerations
For any experimental setup, it is important to consider that the bleaching process and the frequency of acquisition has to match the dynamics of the protein of interest (Lippincott-Schwartz et al., 2003; Weiss, 2004). The above technical details should be first be tested to achieve the optimal conditions for FRAP experiments of specific proteins or for different subcellular compartments. Bleaching and acquisition conditions can be optimized by doing FRAP in fixed cells. We routinely grow cells on glass bottom dishes and fix using 4% PFA in PBS for 15 min at room temperature. After fixation, PFA solution is replaced by imaging media and the FRAP protocols tested on this set of cells. Following this approach, optimization can be achieved in conditions that match the real experimental setup.
The major aims of these optimization experiments are to:
Determine the best conditions suitable for a fast and efficient photobleaching of molecules in a region of interest that would be used in the real experiments.
Optimize the time-lapse settings for acquisition during pre- and, more importantly, post- bleaching regimes. The main aim is to acquire images without causing photobleaching (< ~5%) of the sample at a given frequency that does not compromise FRAP analysis.
Following the optimization steps, a FRAP test is performed in living cells. There are two important points that needs to be considered that are related to the half time of the observed recovery process (Weiss, 2004). Firstly, if the half time is comparable to the bleaching step, then there is a high chance that recovery is underestimated as bleached molecules can diffuse away from the bleached area during the bleaching step (Weiss, 2004). If so, it is necessary to optimize the bleaching protocol to make this step faster (~< 3 times the half time of recovery). This can be achieved for example, by reducing the area of the region that is wanted to be bleached or, by increasing the laser power and reducing the number of iterations during the bleaching step or, by increasing the number of laser lines activated during the bleaching step or, by reducing the scan speed of the bleaching step at the same time the number of iterations it is also reduced. The conditions mentioned for the bleaching step of E-cadherin and Ect2 are good standard initial conditions to perform FRAP experiments on proteins that exhibits very distinctive dynamics. Secondly, slow post acquisition frames can compromise recovery measurements. As the half time of a FRAP curve is calculated with the information acquired during the first 1.5 half times of the recovery process, confident estimation of FRAP parameters requires that acquisition be fast enough to accurately sample this early period. To satisfy this requirement, increasing scan speed or reducing the area of sampling during pre and postbleaching acquisition can increase the speed of acquisition. This second option was chosen in order to capture the fast dynamics of Ect2 mobility.
After these conditions are set, it is essential to consider that the optimized protocol does not compromise the viability of cells. Normally, UV irradiation causes toxicity, which is evident by changes in the morphology of the cell and membrane blebbing (Frigault et al., 2009). Acquisition of phase contrast or Differential interference contrast (DIC) images before and after FRAP acquisition is a complementary test to assess cell viability. Of note, UV irradiation can cause membrane damage that often results in an unexpectedly high immobile fraction. For this, it has been suggested to perform 2 consecutive FRAP experiments on the same cells and on the same region, in order to determine that recovery occurs even after two consecutive rounds of photobleaching (Lippincott-Schwartz et al., 2003).
Image analysis
E-cadherin Turnover
Image analysis was performed using Image J software. Noise on images was reduced by applying a median filter of 2 pixels radii. As E-cadherin dynamics at the ZA is relatively slow (in our experience, a FRAP experiment takes ~10 min to plateau), it is inevitable that some cell movements and/or drift occur during image acquisition. If these movements really compromise the measurements, then the experiment is discarded. However, those experiments with slight cell movements can be corrected and/or eliminated by aligning consecutive frames using Turbo-reg (http://bigwww.epfl.ch/thevenaz/turboreg/) plug-in of Image J. After that, FRAP profiles were calculated using a ROI marked at the bleached area and use the plug-in FRAP profiler to obtain fluorescence intensity profiles. Fluorescence intensities in the ROI immediately after bleaching (F(0)) were subtracted from fluorescence intensities at all times (F(t)) and results were then normalized to pre-bleaching values (Eq.1, Figure 2A). Results were then imported into Prism software for statistics analysis. Data from 11 replicates (3 independent experiments) were pooled and fluorescence intensity at time points after the bleaching step were fitted to the equation:
(Eq.1)
where F(t), F(-t) and F(0) are the average fluorescence of the ROI at any time, before bleaching and, immediately after bleaching, respectively. Mf is the mobile fraction, t1/2 is the half time of recovery and t is time in seconds. In Prism, this fitting is achieved by using non-linear regression and the exponential one-phase association model using Y0 = 0 and where Mf corresponds to the plateau value. Data then are presented as the average ± SEM and the statistical significance assessed by t-test.
Figure 2. Examples of E-cadherin-GFP and GFP-Ect2 FRAP experiments. A. Left, Representative images using MCF-7 cells of the subcellular distribution of E-cadherin-GFP and GFP-Ect2 expressed in E-cadherin and Ect2 knockdown backgrounds, respectively. Center, details of acquisition frames during pre (shown) and post bleaching (not shown) stages during a FRAP experiment. Right, Fluorescence recovery plots for E-cadherin-GFP (top graph) and GFP-Ect-2 (bottom graph). Note the difference in time scales. B. Details of non-linear regression of GFP-Ect2 recovery plot using either mono-exponential (Eq.1) or double exponential (Eq.2) functions. This shows that a mono exponential function does not adjust properly to the experimental curve.
Ect2 Turnover
Image analysis was also performed using Image J software. It is worth to mentioning that an Ect2 FRAP experiment takes ~1 min, therefore no significant drifts or cell movements were observed. To calculate FRAP profiles, a ROI at the bleached GFP-Ect2 area was marked and its average fluorescence determined at every time point using the measure stack plugin in Image J software. Fluorescence intensities were treated as described above for E-cadherin-GFP to obtain recovery plots and data fitted to the double exponential equation (Figure 2B):
(Eq.2)
F(t) is the average fluorescence of the ROI, Mf is the mobile fraction, ffast and fslow are weighting factors for fast and slow mobile components, and their respective half times and t is time in seconds. In Prism, this fitting is achieved by using non-linear regression and the exponential two-phase association model using Y0 = 0 and where the plateau value corresponds to Mf.
For this case, a numerical solution to obtain the t value at which Fluorescence Recovery = 0.5 was applied to obtain the global half time for Ect2 recovery. This was performed in Matlab (MathWorks, Australia) as follows. Values from fitting can be introduced as:
>> (In the brackets real values are introduced)
And then calculate the global t1/2 using the FRAPtwo function (see below) and the following sentence:
>> t1/2 = fzero(@(t) FRAPtwo(Parameters,t),7);
Data are then presented as the average ± SEM and the statistical significance assessed by t-test.
The following is the description of the Matlab function used for calculation of t1/2.
function [ y ] = FRAPtwo(X,t);
plateau=X(1);
fractionfast=X(2);
Kfast=ln(2)/X(3);
fractionslow=X(4);
Kslow=ln(2)/X(5);
y=plateau*fractionfast*(1-exp(-Kfast*t))+plateau*fractionslow*(1-exp(-Kslow*t))-(plateau/2);
end
Recipes
Sodium butyrate
A 1 M stock solution of Sodium butyrate is prepared in water
Filter sterilized
Stored at 4 °C previous to use
Hexadimethine bromide (polybrene)
A stock solution of polybrene is made by diluting it into water to a final stock concentration of 8 mg/ml
Sterilizing by filtering trough a 0.2 μm filter
Imaging media
Hank’s balanced salt solution supplemented with 10 mM HEPES pH 7.4
5 mM CaCl2
4% Paraformaldehyde in PBS
Prepare by dilution of the stock solution (16% formaldehyde)
Adjust pH to 7 with HCl or NaOH if necessary using pH indicator papers
Aliquot dilutions and store at -20 °C
Acknowledgments
This work was supported by the The Kids Cancer Project of The Oncology Children’s Foundation, The University of Queensland Early Career Grant (2012003354) to GAG. RP is supported by UQI (UQ International) Ph.D. Scholarship and ANZ Trustees Ph.D. Scholarship in Medical Research. Confocal microscopy was performed at the ACRF/IMB Cancer Biology Imaging Centre established with the generous support of the Australian Cancer Research Foundation.
References
Frigault, M. M., Lacoste, J., Swift, J. L. and Brown, C. M. (2009). Live-cell microscopy - tips and tools. J Cell Sci 122(Pt 6): 753-767.
Gomez, G. A. and Daniotti, J. L. (2005). H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafficking of H-Ras to this pericentriolar endocytic compartment. J Biol Chem 280(41): 34997-35010.
Goodson, H. V., Dzurisin, J. S. and Wadsworth, P. (2010). Methods for expressing and analyzing GFP-tubulin and GFP-microtubule-associated proteins. Cold Spring Harb Protoc 2010(9): pdb top85.
Kovacs, E. M., Verma, S., Ali, R. G., Ratheesh, A., Hamilton, N. A., Akhmanova, A. and Yap, A. S. (2011). N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway. Nat Cell Biol 13(8): 934-943.
Lippincott-Schwartz, J., Altan-Bonnet, N. and Patterson, G. H. (2003). Photobleaching and photoactivation: following protein dynamics in living cells. Nat Cell Biol Suppl: S7-14.
Otani, T., Ichii, T., Aono, S. and Takeichi, M. (2006). Cdc42 GEF Tuba regulates the junctional configuration of simple epithelial cells. J Cell Biol 175(1): 135-146.
Priya, R., Yap, A. S. and Gomez, G. A. (2013). E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens. Differentiation.
Ratheesh, A., Gomez, G. A., Priya, R., Verma, S., Kovacs, E. M., Jiang, K., Brown, N. H., Akhmanova, A., Stehbens, S. J. and Yap, A. S. (2012). Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens. Nat Cell Biol 14(8): 818-828.
Reits, E. A. and Neefjes, J. J. (2001). From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol 3(6): E145-147.
Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., Sievers, C., Yang, L., Kopinja, J., Rooney, D. L., Zhang, M., Ihrig, M. M., McManus, M. T., Gertler, F. B., Scott, M. L. and Van Parijs, L. (2003). A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33(3): 401-406.
Smutny, M., Cox, H. L., Leerberg, J. M., Kovacs, E. M., Conti, M. A., Ferguson, C., Hamilton, N. A., Parton, R. G., Adelstein, R. S. and Yap, A. S. (2010). Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens. Nat Cell Biol 12(7): 696-702.
Smutny, M., Wu, S. K., Gomez, G. A., Mangold, S., Yap, A. S. and Hamilton, N. A. (2011). Multicomponent analysis of junctional movements regulated by myosin II isoforms at the epithelial zonula adherens. PLoS One 6(7): e22458.
Trenchi, A., Gomez, G. A. and Daniotti, J. L. (2009). Dual acylation is required for trafficking of growth-associated protein-43 (GAP-43) to endosomal recycling compartment via an Arf6-associated endocytic vesicular pathway. Biochem J 421(3): 357-369.
Vitriol, E. A., Uetrecht, A. C., Shen, F., Jacobson, K. and Bear, J. E. (2007). Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins. Proc Natl Acad Sci U S A 104(16): 6702-6707.
Weiss, M. (2004). Challenges and artifacts in quantitative photobleaching experiments. Traffic 5(9): 662-671.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Priya, R. and Gomez, G. A. (2013). Measurement of Junctional Protein Dynamics Using Fluorescence Recovery After Photobleaching (FRAP). Bio-protocol 3(20): e937. DOI: 10.21769/BioProtoc.937.
Download Citation in RIS Format
Category
Cell Biology > Cell imaging > Fluorescence
Cell Biology > Cell imaging > Confocal microscopy
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Improve Research Reproducibility
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DOI: 10.21769/BioProtoc.938 Views: 9476
Reviewed by: Fanglian HeLin Fang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Medicine Feb 2013
Abstract
Myocardial growth goes from proliferation to hypertrophy during development. The measurement of the relative cell area provides information of cardiomyocyte hypertrophy, which is ideal for studying myocardial development.
Materials and Reagents
Mouse embryos
4-6 μm paraffin sections of embryonic hearts
4% PFA
Paraffin
Xylene (Merck KGaA, catalog number: 8.08691.1000 )
Ethanol (Merck KGaA, catalog number: 1.00983.1000 )
PBS
Wheat germ agglutinin (WGA) conjugates tetramethylrhodamine (Life Technologies, Molecular Probes®, catalog number: W849 ) (1:100)
DAPI (Invitrogen, catalog number: D1306 )
Distilled water
Fluorescence mounting media (Fluoromont-G) (SouthernBiotech, catalog number: 0100-01 )
Equipment
Microtome
Fluorescence microscope
Humid chamber
Software
Image analysis software (Image J)
Procedure
Embryos are fixed in 4% PFA and embedded in paraffin. (Mouse embryos are fixed ON at 4 °C and embedded in paraffin after dehydration with Ethanol and Xylene. E16.5 embryos were dehydrated 45 min ethanol 50%, 45 min ethanol 70%, 45 min ethanol 80%, 45 min ethanol 90%, 45 min ethanol 95%, 30 min ethanol 100%, 30 min ethanol 100%, 30 min xylene and 3 times 1-hour paraffin before orientation. Everything at room temperature but the paraffin which is done in a stove at 65 °C)
Rehydrate the sections (65 °C 20 min, xylene 5 min, xylene 5 min, ethanol 100% 5 min, ethanol 100% 5 min, ethanol 96% 5 min, ethanol 90% 5 min, water 5 min).
Wash with Distilled water 5 minutes three times at room temperature.
Wash with 1x PBS 5 min twice at room temperature.
Incubate WGA (1:100) 45 min at room temperature in humid chamber.
Wash with 1x PBS 5 min three times.
Incubate with DAPI (1:1,000) 10 min at room temperature in humid chamber and in darkness.
Wash with 1x PBS 5 min twice.
Mount with fluorescence mounting media (store at 4 °C in darkness before analysis).
Make pictures under the microscope.
Analyse pictures measuring the cell area using ImageJ. Measure at least 100 cells per section and three sections per embryo. Below image example of compact myocardium with the membranes stained with WGA where the relative area was measured. Myocites are identified ignoring the endocardial and endothelial cells, these cells form a monolayer covering the cardiomyocites. Cells within the myocardial wall are measured.
Figure 1. 7 µm transverse sections of a E15.5 WT heart stained with fluorophore-coupled wheat-germ agglutinin (WGA)
Acknowledgments
This study was funded by grants SAF2010-17555, RD06/0014/0038 (RECAVA) and, RD06/0010/1013 (TERCEL) from the Spanish Ministry of Economy and Competition (MINECO) and EU FP7-ITN 215761 (NotchIT) to J.L.d.l.P. G.L. had a PhD fellowship from the MINECO (FPI Program, BES-2008-002904).
References
Luxan, G., Casanova, J. C., Martinez-Poveda, B., Prados, B., D'Amato, G., MacGrogan, D., Gonzalez-Rajal, A., Dobarro, D., Torroja, C., Martinez, F., Izquierdo-Garcia, J. L., Fernandez-Friera, L., Sabater-Molina, M., Kong, Y. Y., Pizarro, G., Ibanez, B., Medrano, C., Garcia-Pavia, P., Gimeno, J. R., Monserrat, L., Jimenez-Borreguero, L. J. and de la Pompa, J. L. (2013). Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy. Nat Med 19(2): 193-201.
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939 | https://bio-protocol.org/en/bpdetail?id=939&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation of Whole Mononuclear Cells from Peripheral Blood
BM Beatriz Martínez-Poveda
GL Guillermo Luxán
JP José Luis de la Pompa
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.939 Views: 7916
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Original Research Article:
The authors used this protocol in Nature Medicine Feb 2013
Abstract
Whole mononuclear cells from peripheral blood are an easy to obtain and useful population of cells where protein and expression patterns of genes can be studied in patients.
Materials and Reagents
EDTA blood (extracted blood kept in tubes with EDTA for anticoagulant purposes)
PBS
Ficoll-Hypaque solution (density 1.077 g/L, Ficoll-Paque PLUS) (General Electric Company, catalog number: 17-1440-03 )
FBS
Complete RPMI-10 (RPMI, 10% FBS)
Equipment
15- or 50 ml conical centrifuge tube
Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent temperature controlled centrifuge)
Procedure
Place fresh EDTA blood into 15- or 50 ml conical centrifuge tube. Using a sterile pipet, add an equal volume of room temperature PBS. Mix well.
Set a layer of the Ficoll-Hypaque solution at the bottom of the centrifuge tube. Use 3 ml Ficoll-Hypaque per 10 ml blood/PBS mixture.
Slowly layer the blood/PBS over the Ficoll at room temperature. Pipette very slowly the mixture against the wall of the tube so that it does not mix with the Ficoll layer.
Centrifuge 30 min in a GH-3.7 rotor at 900 x g at room temperature, with NO brake.
With a pipette remove the upper layer that contains the plasma and most of the platelets. Transfer the aggregates of mononuclear cells to another centrifuge tube (the intermediate layer contains aggregates of cells presenting white colour and floating over the Ficoll).
Wash the mononuclear cells three times with PBS. Add three times the volume of mononuclear cell layer of PBS. Centrifuge 10 min at 400 x g at room temperature.
Resuspend cells in complete RPMI-10 (RPMI, 10% FBS) (10 ml for plating or 1 ml 10% DMSO for freezing). Freeze or plate the cells.
Acknowledgments
This study was funded by grants SAF2010-17555, RD06/0014/0038 (RECAVA) and, RD06/0010/1013 (TERCEL) from the Spanish Ministry of Economy and Competition (MINECO) and EU FP7-ITN 215761 (NotchIT) to J.L.d.l.P. G.L. had a PhD fellowship from the MINECO (FPI Program, BES-2008-002904).
References
John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, and Warren Strober (eds.) (2004). Curr Protocols Immunol. John Wiley & Sons, Inc.
Luxan, G., Casanova, J. C., Martinez-Poveda, B., Prados, B., D'Amato, G., MacGrogan, D., Gonzalez-Rajal, A., Dobarro, D., Torroja, C., Martinez, F., Izquierdo-Garcia, J. L., Fernandez-Friera, L., Sabater-Molina, M., Kong, Y. Y., Pizarro, G., Ibanez, B., Medrano, C., Garcia-Pavia, P., Gimeno, J. R., Monserrat, L., Jimenez-Borreguero, L. J. and de la Pompa, J. L. (2013). Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy. Nat Med 19(2): 193-201.
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Cell Biology > Cell isolation and culture > Cell isolation
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94 | https://bio-protocol.org/en/bpdetail?id=94&type=0 | # Bio-Protocol Content
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Peer-reviewed
Collecting and Fixing Nuclear GFP/RFP in L1 Larva for Imaging
Xiao Liu
Published: Vol 2, Iss 5, Mar 5, 2012
DOI: 10.21769/BioProtoc.94 Views: 10676
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Abstract
In this protocol, L1 stage larvae are collected that carry nuclear-localized GFP/wCherry reporters. These can be fixed so that the GFP/wCherry maintains nuclear localization and stain nuclei by DAPI. This protocol therefore achieves the collection and fixation of nuclei in worm L1 larvae.
Materials and Reagents
Acetone
Formaldehyde
DAPI
Poly-lysine
Glycerol
Cytoseal 280 (Richard-Allan Scientific, catalog number: 8311-4 )
Used Qiaquick Spin Column (QIAGEN)
11.58 μm glassbeads (Whitehouse Scientific, catalog number: MS0012 )
KCl
NaCl
Na2EGTA
Triton X-100
EDTA
PIPES
2x modified MRWB (see Recipes)
DAPI staining solution (see Recipes)
M9 buffer (see Recipes)
Tris triton buffer (TTB) (see Recipes)
Equipment
Standard bench top microcentrifuge
16-slide glass staining jar (Thermo Fisher Scientific, catalog number: 08-810 )
Spatula
Microscope
Glass container
Glass coverslip
Glass slide
18 x 18 mm glass cover slip
25 x 75 mm glass slide
Procedure
Preparing larva
Begin with a plate that contains many eggs (100+)
Use the spatula to remove any chunks which many retain worms. Be sure to flame between uses so no worms are transferred between plates.
Using a spatula, carefully displace and remove the agar from the plastic plate. Place the agar in a 16-slide glass-staining jar keeping the surface with worms facing upwards.
Rinse the agar in the glass container three times with deionized water, taking care that water does not directly hit the agar.
Using the spatula, place the agar back into the plastic container. Remember which side faces upward!
Check under the microscope to make sure no worms are left on the plate. If worms are left, repeat rinses until no worms are left. There should be plenty of eggs (100+).
Leave the plate at room temperature (RT) (25 °C) for 2 h.
Check that L1 larva has emerged.
Freezing and Fixing the Worms
Wash the plate with 500 μl M9 buffer and transfer to 1.5 ml centrifuge tube. Repeat twice.
Spin 3,000 rpm, 2 min. Remove supernatant taking care not to disturb worms at the bottom. Resuspend with 1 ml M9.
Spin 3,000 rpm, 2 min. Remove supernatant taking care not to disturb worms at the bottom. Resuspend with 500 μl M9.
Transfer to a used Qiaquick Spin Column. Spin 3,000 rpm, 2 min with lid open.
Close Qiaquick column cap and place column and collection tube separated into a bucket. Add liquid nitrogen. The next few steps should be performed as quickly as possible after liquid nitrogen is added.
Add 200 μl acetone (-20 °C) to the column and immediately spin 2,000 rpm, 30 sec.
Add 200 μl acetone (-20 °C) to the column and place in -20 °C freezer for 1 min. Then spin 2,000 rpm 30 sec.
Add 200 μl fresh MWRB/formaldehyde solution (50% 2x modified MRWB, 5% formaldehyde = 100 μl 2x modified MRWB, 100 μl 10% formaldehyde) and let sit at RT for 1 h. Then spin at 2,000 rpm, 30 sec.
Add 200 μl TTB and spin 2,000 rpm, 30 sec. Repeat to remove all formaldehyde.
Add 200 μl DAPI staining solution. Let sit at RT for 1 h. Spin 2,000 rpm, 30 sec.
Add 200 μl TTB and pipette up and down to resuspend the worms in the solution. Transfer to 1.5 ml centrifuge tube to collect worms.
Preparing the slides
Add 75 μl of .5% poly-lysine (in H2O) to a 18 x 18 mm glass cover slip. Cover using plastic dish lid and let sit at RT for at least 30 min.
Recollect excess poly-lysine.
Wash cover slip using distilled H2O and air dry.
Add a drop of well suspended 11.58 μm glass beads (in acetone) onto the treated surface of a 25 x 75 mm glass slide. Air dry.
Mounting the Worms
Add 20 μl of worms in TTB to the poly-lysine treated side of the coverslip. Leave at RT for 30 min to allow worms to stick to the coverslip.
Remove as much TTB as possible but observe this removal step under the microscope to make sure most worms are stuck to the poly-lysine.
Add 75 μl 50% glycerol to the coverslip. Carefully remove glycerol from the sides. Add 50 μl 50% glycerol to the middle of the coverslip. This helps to disperse an excess TTB, making the drop closer to 50% glycerol. Remove excess liquid from the sides.
Place slide over the cover slip and seal with Cytoseal 280.
Place in a slide holder and refrigerate until use.
Recipes
2x modified MRWB
160 mM KCl
40 mM NaCl
20 mM Na2EGTA
10 mM Spermidine HCl
30 mM PIPES (pH 7.4)
TTB (Tris triton buffer)
100 mM Tris-HCl (pH 7.4)
1% Triton X-100
1 mM EDTA
M9 buffer
Refer to: common worm media and buffers
DAPI staining solution
100 μl M9
100 μl deionized H2O
0.1 μl 1 mg/ml DAPI
Acknowledgments
This protocol has been adapted from Rigaut et al. (1999) and Puig et al. (2001).
References
Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24(3): 218-229.
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17(10): 1030-1032.
Article Information
Copyright
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Liu, X. (2012). Collecting and Fixing Nuclear GFP/RFP in L1 Larva for Imaging. Bio-protocol 2(5): e94. DOI: 10.21769/BioProtoc.94.
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Category
Cell Biology > Cell imaging > Fluorescence
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940 | https://bio-protocol.org/en/bpdetail?id=940&type=0 | # Bio-Protocol Content
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Peer-reviewed
A High Resolution Short Interfering RNA (siRNA) Detection Method from Virus-infected Plants
Vinay Panwar
Guus Bakkeren
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.940 Views: 12437
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Plant Molecular Biology Apr 2013
Abstract
Plant viruses are strong inducers as well as targets of RNA silencing. In plants RNA silencing acts as a natural defense mechanism against viral infection and is associated with accumulation of virus-specific small interfering RNAs (siRNAs). The continuing discoveries, increasing awareness and interest in the regulatory roles of non-coding small RNAs have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Northern blot analysis of small RNAs involving the separation of RNA molecules using polyacrylamide gel electrophoresis (PAGE) has remained a popular and valuable analytical method to validate small RNAs. Northern blot analysis consist of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization using radioactive probes. The following protocol provides a method for isolation and detection of small RNAs from virus-infected plants and was successfully used in Panwar et al. (2013a), Panwar et al. (2013b).
Keywords: RNA Silencing SiRNA Detection Northern Blotting Virus-Induced Gene Silencing Host-Induced Gene Silencing
Materials and Reagents
Virus-infected plant tissue
TRIzol reagent (Invitrogen)
Chloroform (Sigma-Aldrich)
Isopropanol (Sigma-Aldrich)
Ethyl alcohol (EtOH)
Diethylpyrocarbonate (DEPC) (Sigma-Aldrich)
Hybond NX Neutral Membrane (Amersham biosciences, catalog number: RPN203T )
40% Acrylamide/N’N’-bis-methylene-acrylamide (19:1) (Life Technologies, Ambion®)
Tetramethylethylenediamine (EDTA) (Sigma-Aldrich)
Urea (Sigma-Aldrich)
Hyperfilm TM MP (Amersham biosciences, catalog number: 28-9068-45 )
Ethidium bromide (Sigma-Aldrich)
Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma-Aldrich, catalog number: 39391 )
Ammonium persulphate (APS)
Tetramethylethylenediamine (TEMED) (Sigma-Aldrich)
Tris base (AMRESCO)
Boric acid (Fisher Scientific)
Formamide (Sigma-Aldrich)
Bromophenol blue (Sigma-Aldrich)
Xylene cyanol (Sigma-Aldrich)
3 MM Whatman filter paper
1-methylimidazole (Sigma-Aldrich)
Hydrochloric acid (HCl)
Sodium dodecyl sulphate (SDS) (Fisher Scientific)
20x Saline sodium citrate (SSC) buffer (Sigma-Aldrich)
ULTRAhyb-Oligo buffer (Life Technologies, Ambion®, catalog number: AM8669 )
Megaprime DNA Labeling System (General Electric Company, model: RPN1604 )
Liquid nitrogen
RNase-free water
Small gels (size of regular protein gels 1.5 mm thick) (Bio-Rad, Mini-Protean® Cell)
Neutral Hybond nitrocellulose membrane
RNase ZAP (Life Technologies, catalog number: AM9784 )
DEPC treated water (see Recipes)
10x Tris-Borate-EDTA (TBE) buffer (see Recipes)
10% APS (see Recipes)
15% polyacrylamide gel (see Recipes)
2x loading buffer (see Recipes)
Equipment
Pestle and mortars
Semi dry electroblotter (Bio-Rad)
Protean II vertical gel system (Bio-Rad)
Nanodrop spectrophotometer
Centrifuge
Hybridization oven
UV transilluminator
Microcentrifuge tube
Electrophoresis apparatus
X-ray film
Procedure
RNA isolation
Using mortar and pestle grind 50-100 mg of virus-infected plant tissue in liquid nitrogen. Immediately add 1 ml of TRIzol reagent and continue grinding until a uniform paste is formed. Pipet the homogenized material in to a sterile microcentrifuge tube and incubate for 5 min at 25-30 °C to permit complete dissociation of nucleoprotein complexes.
Add 200 μl of chloroform per ml of TRIzol reagent used for homogenization of sample tissue. Shake the samples vigorously by inverting the microcentrifuge tubes several times by hand and incubate at 20-30 °C for 2-3 min.
Centrifuge the samples at no more than 12,000 x g for 15 min at room temperature (20-25 °C).
Following centrifugation the mixture will separate in a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase.
Without disturbing the interphase, carefully pipette out the aqueous phase and transfer it to a new sterile microcentrifuge tube.
Precipitate the RNA from the aqueous phase by adding 0.5 ml of isopropanol per ml of TRIzol reagent used.
Incubate sample at 20-30 °C for 10 min. Recover the total RNA by centrifuging the sample at no more than 12,000 x g for 10 min at 2-8 °C. RNA will form a gel like pellet at the bottom and side of the microcentrifuge tube.
Without disturbing the pellet remove the supernatant. Wash the RNA pellet by slowly adding 1 ml of 75% ethanol down the side of the tube.
Mix the sample briefly by vortexing and centrifuge at no more than 7,500 x g for 5 min at 2-8 °C.
Discard the supernatant carefully without disturbing the pellet. Vacuum or air dry the pellet for 10-15 min and resuspend in 50 μl RNase-free water (commercially available or DEPC-treated sterile water). The volume is adjustable but higher concentrations are preferable since this will allow lower volumes to be loaded on the gel. Do not overdry the pellet otherwise it will be hard to dissolve. Quantify the concentration of RNA by measuring the absorbance at 260 nm using a spectrophotometer (e.g., Nanodrop) and store at -20 °C until ready to use. RNA will be stable for months at these conditions.
15% Polyacrylamide gel electrophoresis (PAGE)
Small gels (size of regular protein gels 1.5 mm thick) are good to detect the expression of small RNAs. However, to analyze length variants of individual small RNAs (e.g. 20 nt, 21 nt, 22 nt), you need to run longer gels. Before assembling the electrophoresis system, clean all equipment’s thoroughly for any RNase contamination using RNase ZAP. Avoid nuclease contamination throughout the procedure by using sterile solutions and RNase free plasticware.
Assemble gel casting chamber following manufacturer instructions and handcast 15% polyacrylamide gel. Let the gel polymerize for at least 30 min to 1 h at room temperature (20-25 °C). Setup the electrophoresis module as recommended by the manufacturer. Rinse the wells thoroughly with running buffer (0.5x TBE) using a syringe to remove any traces of urea and pre run gel with tracking dye for nearly 15 min before loading the samples.
While pre-running the gel, prepare RNA samples. Ideally, for a good siRNAs resolution, 35-40 μg of total RNA is recommended for loading (based on the purity and concentration of the RNA). Accordingly, adjust the volume of each sample to be loaded (according to the least concentrated sample) using DEPC-treated water. Add equal volumes of 2x loading dye to the samples. Mix well by gentle tapping and spin down briefly.
Cap the tubes and denature RNA samples by heating at 90-100 °C for 15 min. Spin down the samples briefly followed by snap cooling on ice for 5 min.
Load the samples and the siRNA size marker and run the gel at 200 V for nearly an hour.
Note: The amount of voltage and duration of the run for the gel depends on the types of power supply and gel-electrophoresis system used. Stop the run when the bromophenol blue dye has migrated to the end of the gel. Do not overrun.
Semi-dry electroblotting of siRNAs onto nylon membrane
Carefully dismantle the electrophoresis apparatus without breaking the gel. Cut the lower portion of the gel containing both tracking dye (xylene cyanol and bromophenol blue) bands. On a 15% polyacrylamide gel, the bromophenol blue dye front runs near 10 nt long RNA. The upper portion of the gel which includes tRNA and 5S RNA is stained with ethidium bromide solution (0.25 μg/ml ethidium bromide in 0.5x TBE buffer) for 10-20 min. Inspect the integrity and equal loading of RNA in the gel using a 360 nm UV transilluminator. The sharpness and clarity of tRNA bands is a good indicator of RNA quality and integrity (Figure 1).
Figure 1. Northern blot revealing small interfering RNA molecules of 23 nucleotides (upper panel). Ethidium bromide-stained rRNA served as loading controls in the gel prior to RNA transfer (lower panel).
To prepare a transfer sandwich, cut 3 MM Whatman filter paper (six pieces) and neutral Hybond nitrocellulose membrane to the size of the gel. Soak the membrane and Whatman paper in 0.5x TBE buffer until they are wet completely. Complete wetting of the membrane is important to insure proper binding of nucleic acids. Assemble the sandwich as follows.
Place three pieces of Whatman paper on the anode plate of electroblotter. Make sure to roll out any air bubble that may inhibit transfer.
Transfer the membrane on top of the filter papers and roll out any air bubble formed between the membrane and filer paper. After electrophoresis, equilibrate the gel in transfer buffer (0.5x TBE) for 5 to 10 min. Equilibration facilitates the removal of electrophoresis buffer salts and detergents. Carefully place the equilibrated gel over the membrane, aligning the stack as perfect as possible.
Place the remaining three pieces of Whatman paper saturated in running buffer on the stack and roll out any trapped air.
Slightly wet the stack with transfer buffer (0.5x TBE) and place the anode plate on the top without disturbing the gel-nitrocellulose stack and secure firmly.
Note: It is important to exclude excess buffer and air bubbles trapped in the filter paper and membrane when setting up the transfer.
Set the power supply and run the transfer unit for 30 min at 10 V and 200 mAmp (constant current settings). Carefully disassemble the transfer unit and remove the filter paper and gel on top of the nylon membrane. Mark the orientation of the gel slots on the membrane with a pencil on RNA transfer side.
Chemical cross linking
Cross linking of the RNA to the membrane frequently improves the sensitivity of northern blots. RNA can be immobilized to the membrane using conventional methods such as exposure to standard dose of UV (120 mJ/cm2) or by baking in an oven (80 °C for 30 min). Both of these methods can be used successfully, however using the EDC-based cross-linking of small RNAs to membrane greatly improves the signal resolution of small RNAs by hybridization (Pall and Hamilton, 2008).
Immediately prior to use, prepare a solution of 0.16 M EDC solution in 0.13 M 1-methylimidazole and adjust the pH of the solution to 8 with HCl.
Cut a single sheet of 3 MM Whatman filter paper slightly bigger than the size of the membrane and saturate it with the EDC solution.
The membrane is placed on the EDC-saturated 3 MM paper with the side on to which RNA was transferred facing up. Roll out any air bubbles trapped between the membrane and filter paper.
Cover the tray holding the membrane and filter paper with saran wrap and incubate at 60 °C for between 1 to 2 h.
Remove the membrane and rinse with RNase free water to remove any residual EDC solution.
The membrane can be dried and stored at -20 °C after removal of residual EDC without compromising the sensitivity of siRNA detection, or be used immediately for hybridization.
Hybridization
Finally, the membranes with fixed RNAs are incubated with specific, radiolabelled probes. For this, follow general prehybridization and hybridization procedures.
Carry out prehybridization using ULTRAhyb-Oligo buffer for 30 min to one hour at 42 °C with gentle agitation.
Note: Probe signal strength obtained for small RNAs may vary depending on the composition of the hybridization buffer used.
End-label probes with 32P; a number of kits are commercially available but we used the Megaprime DNA labeling system and followed the manufacturer’s instructions. Hybridize the membrane with the prepared 32P-end-labelled oligonucleotide probes. The probes must be labeled at high specific activity (≥ 108 cpm/μg template).
Hybridize the membrane overnight (14-16 h) at 38 to 42 °C with gentle agitation.
Posthybridization, the membrane is washed twice with a low stringency buffer solution (2x SSC, 0.5% SDS) for 10 min each, and once using a high stringency buffer solution (0.1x SSC, 0.1% SDS) for 5 min at 42 °C. If necessary, washing time can be increased under more stringent conditions (e.g., when high background levels are seen).
Expose the membrane to X-ray film at -80 °C for signal visualization. Adjust the exposure time depending on signal intensity.
Note: It is extremely important that all steps involving radioactive material are followed under appropriate safety guidelines.
Recipes
DEPC treated water
Dissolve 1 ml of DEPC in 1 L of distilled water with continuous stirring and autoclave
10x TBE (1 liter)
108 g Tris Base
55 g Boric acid
40 ml EDTA (0.5 M, pH 8.0)
Bring volume to 1 liter with DEPC-treated water, mix by stirring and autoclave
10% APS
Mix 1 g APS in 10 ml of RNase free water and filter sterilize
15% polyacrylamide gel
21 g urea
5 ml 10x TBE
18.8 ml 40% Acrylamide/Bis-acrylamide (19:1)
Bring volume to 50 ml with RNase free water and vortex well until urea is dissolved
When ready to pour gel add by swirling briefly
250 μl 10% Ammonium persulfate
50 μl TEMED
Note: TEMED should be added last. Mix the solution well, immediately pour gel smoothly without creating any air bubbles and let it polymerize. This recipe is sufficient to fill four mini gel cassettes. Amount may be adjusted depending on the application. Gel can be stored in 4 °C degree fridge after tightly wrapping in saran wrap with comb still inserted. Do not freeze gels.
2x loading buffer (50 ml)
47.5 ml 95% Formamide
2 ml EDTA (20 mM, pH 8.0)
0.025 g Bromophenol blue
0.025 g Xylene cyanol
Acknowledgments
This protocol was adapted from the protocol described by Pall and Hamilton (2008). We are grateful for discussions with and technical advice from Mrs. Melanie Walker and Dr. Hélène Sanfaҫon.
References
Pall, G. S. and Hamilton, A. J. (2008). Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3(6): 1077-1084.
Panwar, V., McCallum, B. and Bakkeren, G. (2013a). Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus. Plant Mol Biol 81(6): 595-608.
Panwar, V., McCallum, B. and Bakkeren, G. (2013b). Endogenous silencing of Puccinia triticina pathogenicity genes through in planta-expressed sequences leads to the suppression of rust diseases on wheat. Plant J 73: 521-532.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Panwar, V. and Bakkeren, G. (2013). A High Resolution Short Interfering RNA (siRNA) Detection Method from Virus-infected Plants. Bio-protocol 3(20): e940. DOI: 10.21769/BioProtoc.940.
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Category
Plant Science > Plant molecular biology > RNA > RNA interference
Plant Science > Plant immunity > Perception and signaling
Molecular Biology > RNA > RNA interference
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941 | https://bio-protocol.org/en/bpdetail?id=941&type=0 | # Bio-Protocol Content
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Fluorescence Recovery After Photobleaching (FRAP) in the Fission Yeast Nucleus
Petrina Delivani
MC Mariola R. Chacón
BS Britta Schroth-Diez
Iva M. Tolić-Nørrelykke
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.941 Views: 10318
Reviewed by: Lin FangFanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Cell Biology Jan 2013
Abstract
We use fluorescence recovery after photobleaching (FRAP) to calculate the diffusion coefficient of GFP in the nucleoplasm of fission yeast. The FRAP method can be generally used to measure the mobility of proteins inside the cell or its organelles.
In our experiment we only measured the diffusion of GFP inside the nucleoplasm of fission yeast mitotic cells. However, if GFP is fused to a protein, the mobility of the protein of interest can be calculated following the GFP signal in the bleached area. We did not, however, address this in our experiments; therefore other sources could be searched for this topic.
To compare FRAP and FLIP, both techniques can be used to measure protein mobility inside a cell. However, with FRAP, the diffusion of a protein is measured in the region of interest (ROI), to observe the recovering of fluorescence in this area. In FLIP, fluorescence recovery is measured in an area different from where the bleaching was done, to observe whether the tagged protein is able to move into that area, which would become darker, gaining the bleached proteins. The major difference here is that for FRAP a single bleaching event is sufficient, while FLIP requires a number of bleaching steps, in order to avoid reflux of fluorescent protein in the same region.
Technically FRAP in the nucleus und FRAP in the cytoplasm has no difference. However, we measured a difference between the diffusion coefficient inside the nucleus (D = 5.6 ± 2.8 μm2/s) and in the cytoplasm (D = 8.6 ± 2.2 μm2/s). This is due to the different compositions inside these compartments, consisting of differing amounts of proteins, DNA and RNA.
Materials and Reagents
Fission yeast cells expressing GFP in the nucleus (e.g. PD31 from Kalinina et al., 2013)
Lectin (Sigma-Aldrich, catalog number: L2380 )
MgCl2.6H2O
CaCl2.2H2O
KCl
Na2SO4
Pantothenic acid
Nicotinic acid
Inositol
Biotin
Boric acid
MnSO4
ZnSO4.7H2O
FeCl2.6H2O
Molybdic acid
KI
CuSO4.5H2O
Citric acid
Edinburgh minimal medium (EMM) (see Recipes)
Equipment
Glass bottom dish (35 mm dish with a coverslip number = 1.5 and thickness 0.16 – 0.19) (MatTek, catalog number: P35G-1.5-14-C ) covered with Lectin.Spinning disk confocal microscope with a scanner-based FRAP system
We use the Andor Revolution Spinning Disc System (Andor Technology), consisting of a Yokogawa CSU-X1 spinning disk scan head (Yokogawa Electric), which is connected to an Olympus IX81 inverted microscope (OLYMPUS). The microscope is equipped with a Prior ProScanIII xy scanning stage (Prior Scientific, Rockland MA, USA) and an Olympus UPlanSApo 100x/1.4 NA oil objective (OLYMPUS). Excitation for acquisition and bleaching is done using a Sapphire 488 nm solid-state laser (50 mW; Coherent). Laser power is controlled using the acousto-optic tunable filter in the Andor Revolution laser combiner (ALC, Andor Technology). The microscope is equipped with an iXon EM + DU-897 BV back illuminated EMCCD camera, cooled to -80 °C, pre-amp gain 2.4, EM gain 300 (Andor Technology). The resulting xy-pixel size in the images is 129 nm.
Note: Prior to the FRAP experiments, calibration has to be done following the FRAP calibration routine of Andor iQ software using the Andor FRAPPA calibration slide. Briefly, the software guides the user through a series of point-bleach steps during which one has to indicate the bleached point location in the image. This way the scanner bleach position is calibrated with the point position in the image.
BL HC 525/30 (Semrock)
Spectrometer
Software
Andor iQ2 software (Andor Technology)
Fiji (http://fiji.sc/Fiji)
Matlab (MathWorks) software
Procedure
A 5 ml pre-culture of cells is grown in EMM plus supplements over night. The next day, the cells are refreshed in a 20 ml culture until exponential growth, which is done by diluting 1 ml pre-culture with 19 ml fresh EMM (1:20). Cell number can be measured via optical density measurements on a spectrometer. To obtain cells in exponential growth, an OD600 = 0.1 – 0.2 should be achieved. This would approximate to a cell number between 2 x 106 cells/ml – 1 x 107 cells/ml. An OD600 = 1, is linear to the cell number.
Imaging culture dishes with a glass-bottom are covered with 1 μl Lectin (2 mg/ml) and left to dry.
100 μl of cells in EMM from the culture are added to the dish and left to settle for 10 minutes.
Cells, which were stuck to the dish via lectin, are washed once with 2 ml EMM to remove loose/un-attached cells, and 1.5 ml EMM is added.
FRAP Experiments are performed at room temperature in three successive steps (a. b. c. see below) controlled by Andor iQ2 software. Ideally 30 – 50 cells are measured and analysed.
Prebleach: after focusing the cell of interest, a time-lapse of 50 images of a single plane of the nucleus is acquired with 50 ms exposure time with a 488 nm laser, at 10-15% (details see above, emission filter is BL HC 525/30).
Bleach: bleaching is performed on a 2 x 2 square pixel area (inside the nucleus, away from the nucleolus) with 50% of the 488 nm laser, with a dwell time of 1 ms and 2 repeats on each pixel (details above, emission filter is BL HC 525/30).
Postbleach: following the bleaching, imaging is continued as before for 400 times with 50 ms exposure time of 10-15% of the 488 nm laser (emission filter is BL HC 525/30).
Note: It is important to establish the appropriate laser exposure time and intensity before the FRAP experiment in a ‘not saturated fluorescence condition’ (Arbitrary units of fluorescence go from a range of 0 – 255. In order to have non- saturated conditions, the laser has to be set to an average saturation above 0 but below 255) to avoid excessive bleaching and allow fluorescence quantification for further analysis. This might mean that the image quality is rather poor.
Analysis
Briefly, a region of interest (ROI, see Figure 1) with a width of 5 pixels and a length roughly equal to the length of the nucleus, L, is drawn.
Figure 1. FRAP experiments on GFP in the nucleus of fission yeast. From left to right: A scheme and 3 images of a cell expressing NLS-GFP (strain PD31, Kalinina et al., 2013) before photobleaching (-2.95 s), just after photobleaching (0 s), and the subsequent image (0.059 s). The cross in the middle panel marks the center of the bleached region (2 x 2 pixels). A region of interest (ROI, magenta rectangle) was drawn along the nucleus. Next to the images of the cell, a time-lapse sequence of the enlarged ROI in consecutive images shows the recovery of the GFP. The graph on the right shows the temporal decay of the first Fourier mode. Circles indicate data points, the solid line is a 3-parameter fit (C++) to the function A1(t) = A1(0) exp(-π2Dt/L2) + offset. Here, A1 is the amplitude of the first Fourier mode, t is time, D is the diffusion coefficient, and L is the length of the bleached area. Figure was taken and modified from Kalinina et al. 2013.
The intensities inside the ROI on each image of the movie are summed along the short axis of the ROI.
The resulting one-dimensional fluorescence intensity profiles, corresponding to consecutive time points, are used to calculate the temporal decay of the first Fourier mode.
The diffusion coefficient D was calculated from the decay rate of the amplitude of the first Fourier mode A1(t), as described by Elowitz et al. 1999. For this particular example we get a diffusion coefficient D = 3.7 μm2/s.
Recipes
Depending on the strain, the appropriate medium should be used. For imaging the transparent EMM is best (Forsburg and Rhind, 2006)
For 1 liter solution, weigh in the following components:
3 g/L Potassium hydrogen phthallate 14.7 mM
2.2 g/L
Na2HPO4
15.5 mM
5 g/L
NH4Cl
93.5 mM
20 g/L
Glucose
2% w/v
20 ml/L
Salts (see below, stock solutions)
1 ml/L
Vitamins (see below, stock solutions)
0.1 ml/L
Minerals (see below, stock solutions)
225 mg/L
Leucin (for PD31, which is auxotroph for Leucin)
Salts: 50x Stock solution
Amount
Component
Final concentration
52.5 g/L
MgCl2.6H2O
0.26 M
0.735 g/L
CaCl2.2H2O
4.99 mM
50 g/L
KCl
0.67 M
2 g/L
Na2SO4
14.1 mM
Vitamins: 1,000x Stock
1 g/L
Pantothenic acid
4.20 mM
10 g/L
Nicotinic acid
81.2 mM
10 g/L
Inositol
55.5 mM
10 mg/L
Biotin
40.8 μM
Minerals: 10,000x Stock
5 g/L
Boric acid
80.9 mM
4 g/L
MnSO4
23.7 mM
4 g/L
ZnSO4.7H2O
13.9 mM
2 g/L
FeCl2.6H2O
7.40 mM
0.4 g/L
Molybdic acid
2.47 mM
1 g/L
KI
6.02 mM
0.4 g/L
CuSO4.5H2O
1.60 mM
10 g/L
Citric acid
47.6 mM
Note: Taken and modified from: http://www-bcf.usc.edu/~forsburg/media.html.
Acknowledgments
We thank E. Guarino and the Yeast Genetic Resource Center (YGRC, Japan) for strains and plasmids; the Light Microscopy Facility of MPI-CBG (Dresden, Germany) for discussions and advice; the German Research Foundation (DFG) and the Human Frontier Science Program for financial support. M.R.C. was supported by a Marie Curie Intra-European Fellowship. This protocol was adapted from Kalinina et al. (2013).
References
Elowitz, M. B., Surette, M. G., Wolf, P. E., Stock, J. B. and Leibler, S. (1999). Protein mobility in the cytoplasm of Escherichia coli. J Bacteriol 181(1): 197-203.
Forsburg, S. L. and Rhind, N. (2006). Basic methods for fission yeast. Yeast 23(3): 173-183.
Kalinina, I., Nandi, A., Delivani, P., Chacon, M. R., Klemm, A. H., Ramunno-Johnson, D., Krull, A., Lindner, B., Pavin, N. and Tolic-Norrelykke, I. M. (2013). Pivoting of microtubules around the spindle pole accelerates kinetochore capture. Nat Cell Biol 15(1): 82-87.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Delivani, P., Chacón, M. R., Schroth-Diez, B. and Tolić-Nørrelykke, I. M. (2013). Fluorescence Recovery After Photobleaching (FRAP) in the Fission Yeast Nucleus. Bio-protocol 3(20): e941. DOI: 10.21769/BioProtoc.941.
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Category
Cell Biology > Cell imaging > Fluorescence
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942 | https://bio-protocol.org/en/bpdetail?id=942&type=0 | # Bio-Protocol Content
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Immunolabelling of Thin Slices of Mouse Descending Colon and Jejunum
JB Julien Bellis
ID Isabelle Duluc
JF Jean-Noël Freund
Jan R. De Mey
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.942 Views: 9288
Reviewed by: Lin Fang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Aug 2012
Abstract
This protocol describes a method for efficient immunolabelling of thin tissue slices containing a few rows of intact intestinal crypts, which yields large numbers of them being oriented favorably for recording stacks of optical sections aligned with the crypt long axis (Bellis et al., 2012). The latter can then be used for cell positional analysis, 3D-reconstruction and -analysis. The simple epithelium lining the small intestine is organized into contiguous crypts of Lieberkühn (Potten, 1998; Barker et al., 2012; De Mey and Freund, 2013) several of which making up a crypt/villus unit. Each crypt is a multicellular proliferation unit with a tight hierarchical organization. Under steady state conditions, the epithelium is continuously and rapidly renewed, driven by divisions of multipotent intestinal SCs near the crypt base and cell removal from the villus tip. Techniques for analyzing the organization of the crypts play an important role in the field. Maximal efficiency is obtained by using optical sections obtained from confocal scanning and/or Nomarski optics passing through the center of the longitudinal crypt axis to view the crypt as two cell columns of hierarchical lineage starting from cells positioned at or near the crypt base. This enables positional analysis of certain cellular capacities like performing DNA synthesis, undergoing mitosis and apoptosis (Caldwell et al., 2007; Fleming et al., 2007; Quyn et al., 2010), responding to injury (Potten et al., 1997), or expressing genes (Barker et al., 2012; Bjerknes et al., 2012; Itzkovitz et al., 2012). Our protocol has allowed us to demonstrate that some divisions are asymmetric with respect to cell fate and the occurrence of oriented cell division (OCD) in 80% of the proliferating cells in the upper stem cell and transit amplifying zones. It has further revealed planar cell polarities which are important for crypt homeostasis and stem cell biology and alterations in apparently normal crypts and microadenomas of mice carrying germline Apc mutations shedding new light on the first stages of progression towards colorectal cancer.
Materials and Reagents
Mouse
Isoflurane gas (Abbott Laboratories)
Dissection pan wax (black) (Fisher Scientific, catalog number: S17432 )
Pipes
HEPES
EGTA
MgSO4
Triton-X-100
Taxol (Paclitaxel from Sigma-Aldrich, catalog number: T7191 )
PBS
NaN3
Sodium deoxycholate
BSA 10% in PBS
Tri-sodium citrate dihydrate (Merck KGaA, catalog number: 567446 )
1 N HCl (1 mol/L) (Merck KGaA, catalog number: 1090571000 )
Paraformaldehyde (PFA) 16% solution, EM grade (Electron Microscopy Sciences, catalog number: 15710 )
Phalloidin-Alexa 568 (Life Technologies, Molecular Probes®, catalog number: 12380 )
Note: The Alexa type may be chosen according to available other fluorophores.
DAPI: 1 mg/ml stock solution in PBS (stored at 4 °C)
Alexa-labeled secondary antibodies. In our study, these were from Life Technologies- Molecular Probes goats anti (species) IgG (H + L) and all highly cross-adsorbed to assure absence of cross-species-reactivity.
Note: For any combination of primary antibodies, a choice of corresponding secondary antibodies and fluorophore wavelengths has to be made. When as in our case, Phalloidin-Alexa 568 (red fluorescence) is used for marking microfilaments made of actin, and DAPI for DNA, secondary antibodies are usually labelled with Alexa 488 (green fluorescence) and Alexa 647 (far-red fluorescence). The latter is recommended for antigens giving weak signals, since it is very well detected by current confocal microscopy systems. When Phalloidin could not be used, Alexa 568 or 555 (red fluorescence) labelled secondary antibodies could be used in addition.
Note: In our study, no commercial primary antibodies were used.
Prolong Gold mounting medium without DAPI (Life Technologies, catalog number: P36934 )
2x PHEM (see Recipes)
Fixative (see Recipes)
20 mM Sodium citrate buffer (see Recipes)
Solutions of primary affinity-purified antibodies (see Recipes)
Solutions of secondary antibodies (see Recipes)
Equipment
Thermostatic water bath
Tem Sega evaporator apparatus for isoflurane anesthesia
Scissors 3 cm and microscissors (Fine Science Tools)
Forceps n°5 (Fine Science Tools)
Binocular
30 G 1/2” needles
U-100 Insulin syringe + needle
Microscope slides 76 x 26 x 1.1 mm
1.5 ml Eppendorf tubes
Micropipettes
18 x 18 mm glass coverslips Nr. 1
Fast confocal microscope (we use a Leica SP5 confocal microscope)
63x NA 1.4 oil immersion lens
Procedure
Sample preparation
The mouse needs to be anesthetized during tissue collection. 10 min before anesthetizing it, prepare 15 ml PHEM 1x (from PHEM 2x) in a 50 ml plastic tube and warm it to 37 °C in a thermostatic water bath. Transport the tube in a recipient filled with water at 37 °C to the dissection room and use within minutes.
Anesthetize the mouse using isoflurane inhalation with the help of the Tem Sega evaporator.
While continuing anesthetizing, place it under a binocular microscope placed in a chemical extraction hood, so that the operator does not inhale isoflurane and formaldehyde gases.
Open the abdomen with fine scissors, localize the distal (descending) colon with respect to the anus, free it from surrounding tissue and transect it about 2.5 cm from the latter. For the jejunum, transect it about 2.5 cm from the caecum/proximal colon. For a scheme of the mouse digestive tract, see: http://www.informatics.jax.org/cookbook/figures/figure76.shtml.
Note: For our study, preservation of sensitive cytoskeleton-based structures like mitotic spindles was essential and could only be achieved by rapid handling. We therefore used one animal per intestinal segment and did not straighten out the intestine. For other studies, for example the presence of certain transcription factors in the nucleus of certain cell types, this is probably less critical.
Using a syringe, flush the colon or jejunum section with PHEM 1x at 37 °C.
Note: 37 °C is important for avoiding changes to mitotic spindles that are temperature sensitive. For other studies, this may be less critical.
Flush it immediately with fixative supplemented with 15 μM taxol.
Note: The fixative contains PHEM buffer, known to optimize the preservation of Ca2+ sensitive microtubules (Schliwa and van Blerkom, 1981) and some Triton-X-100 to accelerate the speed of fixation. It is supplemented with taxol, to further prevent microtubule loss. Taxol indeed binds to microtubules and makes them resistant to degradation by formaldehyde fixation. Without added taxol, spindles were found to be shorter and sometimes distorted, making the measurement of spindle angles as described in our study unreliable. When preservation of microtubules is not essential, taxol may be left out. PHEM buffer, however, is recommended since it prevents pH changes during formaldehyde fixation better then for example PBS.
Cut out a segment of 1-2 cm and place it in a cup filled with hardened dissection pan wax (black) filled with 5 ml of fixative at room temperature. Open the colon or jejunum segments with fine scissors by cutting them along their length.
Sacrifice the mouse by cutting through the septum into the heart with scissors.
Pin the tissue flattened and lightly stretched on the wax surface, mucosa up, and continue fixation for 40 min at room temperature.
After about ten minutes, while in fixative, cut the tissue into small 1.5 mm3 cubes, with the help of microscissors.
Transfer these into a 1.5 ml Eppendorf tube and after a total of 40 minutes rinse them three times 10 min in PBS. They can now be stored at 4 °C in PBS supplemented with 8 mM NaN3 for several weeks.
Using a cut-off blue tip on a micropipette, transfer a few pieces onto a microscope slide placed under a binocular. For colon fragments only, remove the muscle lining using two thin 30 G 1/2” needles fixed on a U-100 insulin syringe. To this end, the muscle lining must first be positioned underneath the mucosa layer. They are then separated by inserting one needle between the layers and using it for keeping the muscle layer fixed in place, while using the other to slide or peel the mucosa away.
Note: The muscle lining of the jejunum is thin and fragile, and does not need to be removed.
Using one of the needles as a cutting device while holding in place the mucosa fragment with the other one cut away thin around 1.5 mm long slices containing two to three rows of contiguous crypts. Transfer 30-40 such slices into a 1.5 ml Eppendorf tube. All subsequent steps are performed in such Eppendorf tubes, one per primary antibody combination.
Mount the tubes with about 30 mucosa slices on a rotating wheel and incubate successively during 30 min with 1 ml of PBS containing 200 mM NH4Cl, 3% sodium deoxycholate in H2O, 0.5% Triton-X-100 in PBS and PBS/BSA 1%/Triton 0.2% for blocking. Use a 1 ml blue tip mounted on a micropipette to remove liquids after each incubation step after the slices have sunk by gravity to the tube bottom. Take care to avoid losing slices by holding the tube against a light source.
Note: Preparation of the deoxycholate solution in H2O is imperative.
For labeling with certain antibodies (for example against the transcription factors Atoh1 or Cdx2), perform antigen retrieval by incubating the tubes containing the samples with 1 ml of 0.1 mM sodium citrate buffer, pH 6.0 (prepared from a 20x stock buffer) placed in a block heater at 95 °C for 30 min, before blocking in PBS/BSA 1%/Triton 0.2%.
Immunolabeling of slices
Continue using the same Eppendorf tubes containing slices and mount them on a turning wheel during incubations with primary and secondary antibodies and during washings.
Incubation with first antibodies diluted in 1 ml PBS/BSA 1%/Triton 0.2% is overnight in a cold room at 4 °C.
Rinse 3 x 10 min in 1 ml of PBS/BSA 1%/Triton 0.2%.
Incubate with 1 ml of secondary antibodies diluted in PBS/BSA 1%/Triton 0.2% (2 μl stock antibody supplemented with 2 μl stock Phalloidin-Alexa 568 per tube) for 5 h at RT.
Rinse 2 x 10 min in.
Incubate in 2 μl of stock DAPI diluted in PBS/BSA 1%/Triton 0.2% for 20 min.
Rinse 1x in PBS for 10 min.
Remove the PBS and replace by 150 μl of PBS. Resuspend the slices and reverse the tube to deposit its contents on a microscope slide positioned under the binocular.
Using the U-100 Insulin syringe and needle, collect the slices in the center of the slide.
Using a yellow tip on a micropipette and slightly tilting the slide, remove the PBS to leave the fragments almost dry. The last trace of PBS is removed with the help of a filter paper.
Using a cut-off yellow tip, add 30 μl Prolong Gold mounting medium to the slices and suspend them into it using the U-100 Insulin syringe and needle.
Carefully lower an 18 x 18 mm glass coverslip in order to mount the slices.
Note: About half of the slices will lie on their side.
Leave at RT overnight to allow the mounting medium to polymerize and store in the dark at 4 °C.
Fast confocal microscopy
In order to be able to collect a sufficiently large number of image stacks during one session, we recommend using a fast confocal microscope and a 63x NA 1.4 oil immersion lens. For example, use a Leica SP5 confocal microscope scanning in bidirectional resonance mode (8,000 Hz), with 8x averaging/plane/per channel. Detect DAPI and Alexa 647 simultaneously and Alexa 488 and 568 (or 555) sequentially. Rotate the scanning head so that one field comprises two to three crypts oriented parallel to the scanning direction. An acquisition typically consists of 60 planes x 4 channels, with a step of 0.5 μm and a pixel size of 141 nm.
Numerous images and 3D animations can be consulted in the paper freely downloadable here: http://jcb.rupress.org/articleusage?rid=198/3/331
Recipes
2x PHEM (500 ml)
18.14 g
Pipes
6.5 g
HEPES
3.8 g
EGTA
0.99 g
MgSO4
Dissolve in 500 ml water and adjust to pH 7.0 with 10 mM KOH
Fixative (3% PFA-PHEM 1x-Triton 0.2% -Taxol 15 μM) (10 ml)
1,875 ml
16% PFA
5 ml
2x PHEM
100 μl
20% Triton-X-100
38 μl
4 mM Taxol (dissolved in DMSO)
3 ml
H2O
20 mM Sodium citrate buffer, pH 6.0
Dissolve 5.88 g Tri-sodium citrate dehydrate in 1,000 ml distilled water
Adjust pH to 6.0 with 1 N HCl
Solutions of primary affinity-purified antibodies
Primary affinity-purified antibodies are diluted from stock to 2 to 6 μg antibody/ml in PBS/BSA 1%/Triton 0.2%. Usually, this corresponds to a dilution of 1/500 to 1/2,000.
Note: Each antibody must be tested for specificity and the optimal antibody concentration determined by a serial dilution to achieve between 1 to 6 μg/ml antibody. Ideally, specificity is tested on tissue after suppression of the antigen by knock out or knock down. If the localization is known, obtaining that can suffice.
Solutions of secondary antibodies
Secondary antibodies are used at 2 μl/ml of the stock solution in PBS/BSA 1%/Triton 0.2%, to which 2 μl Phalloidin-Alexa 568 stock solution per ml were added.
Acknowledgments
This protocol has been developed and reported in Bellis et al. (2013). Support was given to J. Bellis by the Ligue Contre le Cancer.
References
Barker, N., van Oudenaarden, A. and Clevers, H. (2012). Identifying the stem cell of the intestinal crypt: strategies and pitfalls. Cell Stem Cell 11(4): 452-460.
Bellis, J., Duluc, I., Romagnolo, B., Perret, C., Faux, M. C., Dujardin, D., Formstone, C., Lightowler, S., Ramsay, R. G., Freund, J. N. and De Mey, J. R. (2012). The tumor suppressor Apc controls planar cell polarities central to gut homeostasis. J Cell Biol 198(3): 331-341.
Bjerknes, M., Khandanpour, C., Moroy, T., Fujiyama, T., Hoshino, M., Klisch, T. J., Ding, Q., Gan, L., Wang, J., Martin, M. G. and Cheng, H. (2012). Origin of the brush cell lineage in the mouse intestinal epithelium. Dev Biol 362(2): 194-218.
Caldwell, C. M., Green, R. A. and Kaplan, K. B. (2007). APC mutations lead to cytokinetic failures in vitro and tetraploid genotypes in Min mice. J Cell Biol 178(7): 1109-1120.
De Mey, J. and Freund, J.-N. (2013). Understanding epithelial homeostasis in the intestine: An old battlefield of ideas, recent breakthroughs, and remaining controversies. Tissue Barriers 1(2): e24965.
Fleming, E. S., Zajac, M., Moschenross, D. M., Montrose, D. C., Rosenberg, D. W., Cowan, A. E. and Tirnauer, J. S. (2007). Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine. J Histochem Cytochem 55(11): 1173-1180.
Itzkovitz, S., Lyubimova, A., Blat, I. C., Maynard, M., van Es, J., Lees, J., Jacks, T., Clevers, H. and van Oudenaarden, A. (2012). Single-molecule transcript counting of stem-cell markers in the mouse intestine. Nat Cell Biol 14(1): 106-114.
Potten, C. S., Booth, C. and Pritchard, D. M. (1997). The intestinal epithelial stem cell: the mucosal governor. Int J Exp Pathol 78(4): 219-243.
Potten, C. S. (1998). Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos Trans R Soc Lond B Biol Sci 353(1370): 821-830.
Quyn, A. J., Appleton, P. L., Carey, F. A., Steele, R. J., Barker, N., Clevers, H., Ridgway, R. A., Sansom, O. J. and Nathke, I. S. (2010). Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue. Cell Stem Cell 6(2): 175-181.
Schliwa, M. and van Blerkom, J. (1981). Structural interaction of cytoskeletal components. J Cell Biol 90(1): 222-235.
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Category
Stem Cell > Adult stem cell > Intestinal stem cell
Biochemistry > Protein > Immunodetection
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Immunoblot Analysis of Histone H4 Acetylation and Histone H2A Phosphorylation in Candida albicans
Michael Tscherner
Karl Kuchler
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.943 Views: 8971
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Microbiology Dec 2012
Abstract
Posttranslational modifications of histones are required for different processes including transcription, replication and DNA damage repair. This protocol describes the preparation of a whole-cell extracts for the fungal pathogen Candida albicans. Furthermore, the extract is used to detect lysine acetylation of histone H4 as well as serine 129 phosphorylation of histone H2A by immunoblot analysis.
Keywords: Histone Acetylation Phosphorylation Candida albicans
Materials and Reagents
Candida albicans
TCA (trichloroacetic acid) (Merck KGaA, catalog number: 641730 )
Recombinant histone H4 (New England Biolabs, catalog number: M2504S )
Calf histones (Sigma-Aldrich, catalog number: H9250 )
0.033% sodium azide (Merck KGaA, catalog number: 8223350250 )
Nitrocellulose membrane (Millipore, catalog number: Protran BA79)
Whatman filter paper 3 MM Chr (Whatman, catalog number: 3030-917 )
BSA (PAA Laboratories GmbH, catalog number: K41-001 )
Sodium azide (Merck KGaA, catalog number: 822335 )
Rabbit polyclonal antibody to histone H4 acetyl K5 (Abcam, catalog number: ab51997 ) (dilution: 1:5,000)
Rabbit polyclonal antibody to histone H4 acetyl K8 (Active Motif, catalog number: 39172 ) (dilution: 1:3,000)
Rabbit polyclonal antibody to histone H4 acetyl K12 (Millipore, catalog number: 07-959 ) (dilution: 1:3,000)
Rabbit polyclonal antibody to histone H4 C-terminus (Abcam, catalog number: ab10158 ) (dilution: 1:1,000)
Rabbit polyclonal antibody to histone H2A phospho-serine 129 (Active Motif, catalog number: 39271 ) (dilution: 1:2,000)
Rabbit polyclonal antibody to histone H2A (Active Motif, catalog number: 39236 ) (dilution: 1:5,000)
IRDye 800CW goat anti-rabbit IgG (H + L) (LI-COR, catalog number: 926-32211 )
IRDye 680RD goat anti-rabbit IgG (H + L) (LI-COR, catalog number: 926-68071 )
4.5% stacking gel
20% running gel
Bacto Yeast Extract (Becton, Dickinson and Company, catalog number: 212720 )
Bacto Peptone (Becton, Dickinson and Company, catalog number: 211820 )
Glucose (Merck KGaA, catalog number: 346351 )
β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148-100ML )
Urea (Sigma-Aldrich, catalog number: U5378-1KG )
SDS (AppliChem GmbH, catalog number: A1112,1000 )
EDTA (Sigma-Aldrich, catalog number: E5134-1KG )
Bromphenolblue
YPD medium (see Recipes)
Yex lysis buffer (see Recipes)
Protein sample buffer (see Recipes)
SDS-PAGE running buffer (see Recipes)
Running buffer (see Recipes)
Transfer buffer (see Recipes)
TBS and TBS-T (see Recipes)
PBS (see Recipes)
Equipment
Mini-Protean gel electrophoresis system (Bio-Rad, model: 165-8000 )
Mini Trans-Blot cell (Bio-Rad, model: 170-3930 )
1.5 ml microcentrifuge tubes
15 ml Falcon tubes
14 ml Snap-cap tubes
Eppendorf Thermomixer comfort (Eppendorf, model: 5355 000.011 )
Shaking incubator
Orbital shaker
LI-COR Odyssey CLx Infrared scanner (LI-COR, model: P/N 9140-WP )
CASY Cell Counter Model TT (Roche, catalog number: 05651735001 )
Software
LI-COR Odyssey CLx imaging system
Procedure
Whole-cell extract preparation
Inoculate a single colony in 5 ml of YPD medium in a 14 ml Snap-cap tube and grow overnight at 30 °C shaking with 220 rpm.
Dilute overnight culture to an OD600 of 0.1 in 5 ml YPD medium in a 14 ml Snap-cap tube and grow cells to OD600 of 1 at 30 °C shaking with 220 rpm.
Note: For strains/conditions with different cell morphologies determine cell number by CASY measurement according to the CASY operator manual and use a total number of 5 x 107 cells. Flasks can also be used instead of Snap-cap tubes.
Harvest cells by centrifugation in 15 ml Falcon tubes for 3 min at 1,500 x g.
Resuspend pellet in 1 ml ice-cold H2O.
Note: Work on ice from now on.
Transfer suspension to 1.5 ml tube.
Add 150 μl ice-cold Yex lysis buffer, vortex thoroughly and incubate on ice for 10 min.
Add 150 μl ice-cold 50% (w/v) TCA and incubate on ice for 10 min to precipitate proteins.
Spin for 5 min at 10,000 x g 4 °C and discard supernatant with a pipette.
Spin for 1 min at 10,000 x g 4 °C and remove rest of the supernatant.
Resuspend pellet in 100 μl protein sample buffer.
Note: Resuspend by pipetting; if pH turns acidic (protein sample buffer turns yellow), add 1 M Tris base to increase pH (until sample buffer turns blue again).
Incubate at 37 °C for 15 min shaking at 900 rpm.
Spin down cell debris at 10,000 x g for 5 min and use 10 μl (0.5 OD600 units) of the supernatant for SDS-PAGE.
SDS-PAGE and western blotting
Load samples on a polyacrylamid gel (4.5% stacking gel and 20% running gel).
Load 0.5 μg recombinant histone H4 in protein sample buffer as negative control and 2 μg calf histones in protein sample buffer as positive control for acetylation analysis.
Note: Gel cast as described previously (Sambrook and Russell, 2001); Bio-Rad Mini-Protean gel electrophoresis system was used.
Run gel at 150 V.
Transfer proteins to nitrocellulose membrane by electroblotting using the Bio-Rad Mini Trans-Blot cell.
Assemble blotting cassette in transfer buffer according to the Mini Trans-Blot instruction manual and run at 200 mA for 1 h at room temperature.
Block membrane for 1 h at room temperature on an orbital shaker using 5% (w/v) BSA in TBS.
Incubate with primary antibody diluted in TBS-T overnight at 4 °C on an orbital shaker.
Pour off primary antibody solution and wash 3 x 10 min in TBS-T at room temperature.
Note: Primary antibody solution can be reused several times (depending on the antibody); if solution is reused, add 0.033% sodium azide as preservative.
Incubate with secondary antibody diluted 1:10,000 in TBS-T for 45 min at room temperature on an orbital shaker.
Note: IRDye 800CW or IRDye 680RD secondary antibodies can be used.
Pour off secondary antibody solution and wash 3 x 10 min in TBS-T at room temperature.
Rinse blot briefly with PBS and scan using the LI-COR Odyssey CLx Infrared scanner.
Note: Scanner settings: Intensity: 5; Resolution: 168 μm; Quality: medium.
Recipes
YPD medium
10 g/L Bacto Yeast Extract
20 g/L Bacto Peptone
20 g/L Glucose (add after autoclaving as 10x stock)
Yex lysis buffer
1.85 M NaOH
7.5% (v/v) β-Mercaptoethanol (freshly added)
Protein sample buffer
40 mM Tris-HCl, pH 6.8
8 M Urea
5% (w/v) SDS
0.1 mM EDTA
1% (v/v) β-Mercaptoethanol (freshly added)
0.1 g/L Bromphenolblue
Running buffer
25 mM Tris
192 mM Glycine
0.1% (w/v) SDS
Transfer buffer
25 mM Tris
192 mM Glycine
20% (v/v) Methanol
TBS
25 mM Tris
140 mM NaCl
2.5 mM KCl
pH 7.4
TBS-T
TBS with 0.1% (v/v) Tween 20
PBS
140 mM NaCl
2.5 mM KCl
8.1 mM Na2HPO4
1.5 mM KH2PO4
pH 7.3
Acknowledgments
We thank all laboratory members for helpful discussions. This work was supported by a grant from the Christian Doppler Society, and in part by a grant from the Austrian Science Foundation (Project FWF-P-25333), to K.K. M.T. was supported through the Vienna Biocenter PhD Programme WK001.
References
Sambrook, J. & Russell, D.W. (2001). Molecular Cloning, Volume 3, 3rd edition, Cold Spring Harbor Laboratory Press.
Tscherner, M., Stappler, E., Hnisz, D. and Kuchler, K. (2012). The histone acetyltransferase Hat1 facilitates DNA damage repair and morphogenesis in Candida albicans. Mol Microbiol 86(5): 1197-1214.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Tscherner, M. and Kuchler, K. (2013). Immunoblot Analysis of Histone H4 Acetylation and Histone H2A Phosphorylation in Candida albicans. Bio-protocol 3(20): e943. DOI: 10.21769/BioProtoc.943.
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Category
Microbiology > Microbial biochemistry > Protein > Immunodetection
Molecular Biology > Protein > Detection
Biochemistry > Protein > Immunodetection > Immunostaining
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[3H]-Spiperone Competition Binding to Dopamine D2, D3 and D4 Receptors
JW Jan-Peter van Wieringen
MM Martin C. Michel
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.944 Views: 8698
Reviewed by: Fanglian HeCheng Zhang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Naunyn-Schmiedeberg's Archives of Pharmacology Dec 2012
Abstract
This protocol is intended for use in 96 well plates (1,200 μl wells) but it can similarly be applied to standard test tubes (Levant, 2007). D2, D3, and D4 dopamine receptors are members of the D2-like class of dopamine receptors. They can be studied using the radioligand [3H]-spiperone, which is an antagonist binding to D2, D3, and D4 receptors with comparable affinity. A competition experiment is usually performed to determine the affinity of a compound for a receptor. If multiple subtypes or states of the receptor are present and the competing compound differentiates them, a competition binding experiment can quantify the relative contribution of the two subtypes or states; while resolution of more than two subtypes or states is theoretically possible, in practical terms it is almost never feasible. Thus, radioligand binding to a receptor is quantified in the presence of various concentrations of the unlabelled compound of interest. The concentration of the radioligand in a competition study should be about 2-3 its Kd value as determined in saturation binding; this will allow a sufficient occupancy of the receptor to obtain a strong signal and at the same time avoid that competition becomes too difficult due to high radioligand concentration. The incubation time and temperature are chosen to allow formation of equilibrium between association and dissociation with the receptor for both radioligand and competitor. Of note, a simple competition experiments does not necessarily prove a competitive nature of the interaction between unlabelled drug and receptor. If the specific radioactivity is low (tritiated) relative to the affinity of the radioligand (< 1 nM), a high assay volume (≥ 1 ml) is required to avoid ligand depletion; this is of particular importance if a receptor source with high expression density is used (e.g. expressed recombinant receptors). The number of required competitor concentrations depends on the goal of the experiment. If only a rough estimate of antagonist potency is required, 1-2 concentration per log increment will be sufficient. However, if it is the aim to test for possible subtypes or states of the receptor, 3-5 concentrations per log increment are needed. If possible, the lowest competitor concentrations in the assays should not cause any detectable inhibition, whereas the highest concentrations should completely abolish specific binding. Each experiment can be divided into different steps such as assay preparation, membrane preparation, incubation, filtration, counting of the samples and data analysis. To minimize experimental error all assays are performed at least in duplicate. Additionally, duplicates of total binding and non-specific binding should be included in the assay; the agent used for the definition of non-specific binding (NSB) should be chemically (different family) and physically (avoid combination of two lipophilic compounds) distinct from the radioligand to avoid artifacts. For discussion of specific benefits of chosen assay conditions see van Wieringen et al., (2013) (copy can be obtained from the author).
Keywords: Dopamine Receptor Radioligand Binding Spiperone
Materials and Reagents
Radioligand [3H]-spiperone (e.g. PerkinElmer, catalog number: NET565250UC )
Butaclamol (e.g. Sigma-Aldrich, catalog number: 55528-07-9 )
Receptor-containing membrane suspension
Competitor compounds (dopamine HCl used as example here, e.g. Sigma-Aldrich, catalog number: 62-31-7 )
Whatman GF/C filters (e.g. PerkinElmer, catalog number: 6005174 )
Poly(ethyleneimine) solution (PEI) (e.g. Sigma-Aldrich, catalog number: 9002-98-6 )
Scintillation cocktail (e.g. PerkinElmer, catalog number: 6013641 )
TRIS base
CaCl2
MgCl2
Distilled water
Ascorbic Acid
Assay buffer (see Recipes)
Wash buffer (see Recipes)
0.1% PEI (only for D3 receptor assay) (see Recipes)
NSB solution (see Recipes)
Dilutions of competitors compound (see Recipes)
Radioligand solution (see Recipes)
Equipment
96 well plates (polysterene)
Cell harvester (e.g. PerkinElmer)
Ultra-Turrax (IKA, model: 0001602800 ) or similar disperser
Water bath
Scintillation counter
Software
Prism (Graphpad Software) or similar
Procedure
Before the assay
Prepare assay and wash buffer.
For a D3 assay: Prepare 0.1% PEI solution and pipet 100 μl/filter on the filterplate, subsequently place in refrigerator (4 °C) for at least 2 hours.
The experiment is performed in 96 well plates (polysterene) with a total assay volume of 1,000 μl (450 μl assay buffer + 200 μl radioligand + 200 μl of assay buffer or 5 μM (+)-butaclamol + 150 μl membrane preparation).
Prepare NSB solution.
Prepare dilutions of competitor compound (dopamine used as example here).
Prepare radioligand solution.
Membrane preparation.
Prepare receptor-containing membrane suspension according to preparation protocol. Re-homogenize suspension in small volume (< 2 ml) using short burst of Ultra-Turrax. Dilute to the desired protein concentration and to yield a total volume of about 8 ml per 48 data point experiment. The protein concentration of the membrane suspension should be chosen so that a robust specific binding signal is obtained but at the same time total binding should be < 10% (even better < 5%) of free radioligand concentration. Protein content can be assayed by a variety of essays, e.g. Bradford (1976). Prepare the membrane suspension initially in ice.
Pre warm all solutions for 15 min in 25 °C waterbath.
Final preparation (Table 1), add components to wells in following order:
250 μl assay buffer.
150 μl membrane suspension.
200 μl competitor, assay buffer (TB) or butaclamol (NSB). Use increasing concentrations of competitor.
200 μl assay buffer.
Start reaction by adding 200 μl radioligand solution.
Table 1. Pipetting scheme for competitor concentrations on microtiter plate
TB
TB
NSB
NSB
10
10
2 x 10
2 x 10
3 x 10
3 x 10
5 x 10
5 x 10
9
9
2 x 9
2 x 9
3 x 9
3 x 9
5 x 9
5 x 9
8
8
2 x 8
2 x 8
3 x 8
3 x 8
5 x 8
5 x 8
7
7
2 x 7
2 x 7
3 x 7
3 x 7
5 x 7
5 x 7
6
6
2 x 6
2 x 6
3 x 6
3 x 6
5 x 6
5 x 6
5
5
NSB
NSB
Total
Total
During the assay
Incubate for 120 min at 25 °C in water bath.
Terminate reaction by rapid vacuum filtration over Whatman GF/C filters using a cell harvester. Wash filter 10 times with ice-cold wash buffer.
After the assay
Add aliquots of 50 μl radioligand to wells of counting plate to determine total radioactivity.
Dry, e.g. in oven for 2 h.
Place sticker on bottom and add scintillation cocktail (20 μl) to filters, place sticker on top of plate and count in a scintillation counter, allow adequate time (15 min) before counting samples.
Data analysis
The data being obtained can be analyzed to yield several types of information. The following should be considered.
Carefully inspect raw data for consistency of replicates and resist the temptation to beautify the data by eliminating apparent ‘outliers’. It is our recommendation that one should very conservative in this regard; the result of a well-designed experiment will not be heavily affected by a single outlier. In other words, if the outcome of the experiment hinges on the question whether a single data point is an outlier, the overall experiment may have been designed and/or executed poorly and should probably be repeated.
Plot amount of binding in the absence and presence of competitor on the y-axis vs. concentration of the competitor on the x-axis. In such plots the amount of binding in the absence of competitor (total binding, TB) can be entered at a virtual concentration lower than any tested competitor concentration, and non-specific binding (NSB) at a virtual concentration higher than the highest tested competitor concentration. A representative experiment with D2 receptors may look like this.
Using all replicates without averaging them is preferable for the data analysis.
In a well performed experiment, the concentration range of the competitor has been chosen in a way that the lowest competitor concentration does not cause any measurable inhibition and that the highest competitor concentration yields a degree of inhibition close to non-specific binding. If one of these two conditions is not met, data analysis becomes tricky as curve fitting may yield spurious values.
Apply one of many available data analysis software packages, e.g. Prism, to fit a sigmoidal curve to the data. In most of such computer programs, you have a choice of settings which have important implications for the interpretation of the resulting parameter estimates.
The preferred option is to let the top and the bottom of the curve to be found be the software, and the resulting values should be checked whether they are close to the experimentally determined total and non-specific binding.
The turning point of the sigmoidal curve should be found by the software in most cases as this will yield the IC50 of the curve.
The slope of the curve can be found by the software. Values close to unity indicate interaction with a single site. If the slope is smaller than unity, i.e. the curve is shallow, this may indicate interaction with multiple sites (see below) but does not necessarily prove it.
If the assumption is made that an interaction with multiple sites, e.g. receptor subtypes, is possible, it can be defined that the curves should follow a monophasic or a biphasic function with each component having a slope of unity. On theoretical grounds a biphasic fit always yields a smaller residual error, as there are more fitting parameters. Therefore, a biphasic fit should only be accepted if it yields a significantly smaller residual error as judged by an F-test or similar (already implemented in most software packages for such use). If a biphasic fits is superior, it will yield IC50 values for both components and a percentage of each of the two components. e.g. for the representative experiment shown above, the sum of squares of distances of data points to the fitted curve was 24177 and 18221 for the one- and two-site fit, respectively, yielding an F value of 6.734, which indicates with a p-value of 0.0040 that the null hypothesis (single site is preferred) should be rejected and a two-site model should be preferred.
The ability to pick up biphasic curves depends on the selectivity of the competitor for the two subtypes being present (or the intrinsic activity of the agonist, if agonist high- and low-affinity states are being analyzed) as well as the number of inhibitor concentrations being tested per log unit increment of concentration.
The results of the curve fitting should always be inspected visually to check whether they make sense. e.g. in some cases the computer program yields estimates which are outside the range of tested competitor concentrations. Such extrapolations are highly unreliable and should not be used; rather the experiment should be repeated with a better choice of competitor concentrations.
Estimates of IC50 depend on the concentration of radioligand in the assay, expressed as fold of its Kd value. To obtain the more informative Ki value, transformation of IC50 is necessary by the Cheng & Prusoff equation:
Ki = IC50/((L/Kd) + 1)
in which L and Kd are the concentration and affinity of the radioligand.
Notes:
A competition binding experiment by virtue of its design cannot prove a competitive interaction between inhibitor and radioligand. This requires e.g. saturation experiments in the absence and presence of one or more inhibitor concentrations.
As IC50 values are obtained from a log scale, the replicates from multiple experiments typically do not exhibit a Gaussian distribution on a linear scale. Hence, the average from multiple experiments should be presented as means (with SD) of –log IC50 (or –log Ki). Alternatively, the median IC50 or Ki can be presented with (asymetric) confidence intervals. Means (with SD) of linear IC50 or Ki values are inappropriate.
Recipes
Assay buffer
50 mM TRIS: TRIS-HCl: 6.6 g and TRIS base 970 mg/L (or only 6.04 g TRIS base/L)
5 mM KCl: 373 mg/L
2 mM CaCl2: 220 mg/L
2 mM MgCl2.6H2O: 410 mg/L
pH 7.4
Every test day prepare a fresh buffer containing 0.05% (25 mg/50 ml) ascorbic acid
Note: This buffer is optimized for detection of agonist high-affinity states of a receptor (agonist competition curves); hence, inclusion of Na+ should be avoided. If that is not within the scope of the project, a buffer without Mg2+, Ca2+ or K+ can be used.
Wash buffer
50 mM TRIS: TRIS-HCl (33 g/5 L) and TRIS base (4.85 g/5 L)
pH 7.4
0.1% PEI (only for D3 receptor assay)
Prepare 0.1% solution, PEI is delivered as a 50% solution, pipet 1 ml from this with a syringe and add to 9 ml aqua dest. to get 5%, pipet 400 μl PEI 5% + 19.6 ml destilled water to get PEI 0.1%
NSB solution
3.98 mg butaclamol HCl/10 ml assay buffer yields 1.10-3 M (prepare aliquots of this)
Dilute this 1:200 to obtain 5 μM in solution, i.e. 1 μM final concentration in asay
Dilutions of competitors compound (dopamine used as example here)
Molecular weight of dopamine HCl is 189.6 g/mol. Weigh 3-5 mg, calculate needed volume of assay buffer to reach target concentration; trying to start with a fixed volume and to reach a specific amount of compound is technically more difficult. In this example 3.8 mg has to be dissolved in 20 ml assay buffer to reach 10-3 M. Dilute this 2x with assay buffer to reach 5x the intended highest concentration in the assay, e.g. 5 x 10-4 M for 10-4 M in the assay.
In the first row of below scheme place 1,800 μl assay buffer per tube, in the 2nd row (2x) 800 μl, in the 3rd row (3x) 700 μl and in the 4th row (5x) 500 μl. Amounts to be prepared may need to be increased if more than one competition experiment is to be performed.
Dilute competitor stock solution in 1:10 steps first. Pipet from 10-4 200 μl to 5 and from 5,200 μl to 6 etc. Then, make 2x, 3x and 5x daughter solutions, pipet 200 μl in 2x, 300 μl in 3x and 500 μl in 5x. Do this also for 8, 7, 6, 5 and 4. (Table 2)
Table 2. Dilution scheme for competitor working solutions
5x
5x
5x
5x
5x
NSB
3x
3x
3x
3x
3x
2x
2x
2x
2x
2x
9
8
7
6
5
4
Radioligand solution
The intended radioligand concentration in the assay should be about 2-3x Kd (Kd spiperone in transfected cells according to literature ≈ 0.05 nM, 0.35 nM and 0.07 nM for D2, D3 and D4, respectively). The stock solution to be prepared needs to be 5x the assay concentration. Thus, the stock solution should be 10-15x Kd. The following is an example calculation based on a specific activity of 16.2 Ci/mmol radioactive of the radioactive stock solution for a D2 receptor assay. This needs to be adapted for the other subtypes based on their Kd values and for each batch of radioligand and its specific activity. Prepare about 10 ml of stock solution per planned 48 data point experiment. Stock solution can be prepared in 50 ml tube with screw cap.
[3H]-spiperone concentration = 1 mCi/ml/(16.2 Ci/mmol x 1,000 mCi/Ci) = 61.7 μM
Highest concentration needed (3 x Kd x 5) = 3 x 0.05 x 5 = 0.75 nM
10 ml x 0.75 nM = 0.0075 nmol [3H]-spiperone needed
(0.0075 nmol)/61.7 x 103 nM = 0.12 μl of [3H]-spiperone solution needed for 10 ml
Thus 1 μl [3H]-spiperone solution in 83.3 ml assay buffer to give a concentration of 0.75 nM
A free tool for such calculations can be found at www.graphpad.com/quickcalcs/chemMenu/.
Acknowledgments
This protocol is the adaptation of a protocol originally published by Levant (2007).
References
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Levant, B. (2007). Characterization of Dopamine Receptors. Curr Protoc Pharmacol 36:1.6.1–1.6.15.
van Wieringen, J. P., Booij, J., Shalgunov, V., Elsinga, P. and Michel, M. C. (2013). Agonist high- and low-affinity states of dopamine D(2) receptors: methods of detection and clinical implications. Naunyn Schmiedebergs Arch Pharmacol 386(2): 135-154.
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© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Wieringen, J. V. and Michel, M. C. (2013). [3H]-Spiperone Competition Binding to Dopamine D2, D3 and D4 Receptors. Bio-protocol 3(20): e944. DOI: 10.21769/BioProtoc.944.
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Biochemistry > Protein > Interaction > Protein-ligand interaction
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945 | https://bio-protocol.org/en/bpdetail?id=945&type=0 | # Bio-Protocol Content
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Peer-reviewed
[3H]-Spiperone Saturation Binding to Dopamine D2, D3 and D4 Receptors
JW Jan-Peter van Wieringen
MM Martin C. Michel
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.945 Views: 10025
Reviewed by: Fanglian HeCheng Zhang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Naunyn-Schmiedeberg's Archives of Pharmacology Dec 2012
Abstract
This protocol is intended for use in 96 well plates (1,200 μl wells) but it can similarly be applied to standard test tubes (Levant, 2007). D2, D3, and D4 dopamine receptors are members of the D2-like class of dopamine receptors. They can be studied using the radioligand [3H]-spiperone, which is an antagonist binding to D2, D3, and D4 receptors with comparable affinity. A saturation assay can be used to determine the affinity of a radioligand to a receptor (Kd) and to determine the total number of receptors present in the assay (Bmax). If saturation binding experiments are performed in the absence and presence of a fixed concentration of another, not radiolabeled ligand, it can also be determined whether the other ligand acts in a competitive manner. If the specific radioactivity is low (tritiated) relative to the affinity of the radioligand (< 1 nM), a high assay volume (≥ 1 ml) is required to avoid ligand depletion; this is of particular importance if a receptor source with high expression density is used (e.g. expressed recombinant receptors). To obtain reliable estimates of these parameters at least 6 different concentrations of radioligand must be tested, but particularly when a receptor is first detected in a given tissue or cell type a greater number of concentrations are helpful. The incubation time and temperature are chosen to allow formation of equilibrium between association and dissociation with the receptor for both radioligand and competitor. Each experiment can be divided into different steps such as assay preparation, membrane preparation, incubation, filtration, counting of the samples and data analysis. To minimize experimental error all assays are performed at least in duplicate. Radioligand dilutions should be prepared to cover the desired concentration range. Optimally these concentrations should cover both the high range (corresponding to 5-10x Kd and hence saturation of the receptor) and the low range (around Kd), so that both Kd and Bmax can reliably be estimated without undue extrapolation. At each radioligand concentration total and non-specific binding should be determined; the agent used for the definition of non-specific binding (NSB) should be chemically (different family) and physically (avoid combination of two lipophilic compounds) distinct from the radioligand to avoid artifacts. For discussion of specific benefits of chosen assay conditions see van Wieringen et al. (2013) (copy can be obtained from the author).
Keywords: Dopamine Receptor Ra
Materials and Reagents
Radioligand [3H]-spiperone (e.g. PerkinElmer, catalog number: NET565250UC )
Butaclamol (e.g. Sigma-Aldrich, catalog number: 55528-07-9)
Receptor-containing membrane suspension
Whatman GF/C filters (e.g. PerkinElmer, catalog number: 6005174 )
Poly(ethyleneimine) solution (PEI) (e.g. Sigma-Aldrich, catalog number: 9002-98-6 )
Scintillation cocktail (e.g. PerkinElmer, catalog number: 6013641 )
Tris-HCl
KCl
CaCl2
MgCl2
Destilled water
Assay buffer (see Recipes)
Wash buffer (see Recipes)
NSB solution (see Recipes)
Radioligand solutions (see Recipes)
Radioligand Dilutions (see Recipes)
Equipment
96 well plates (polysterene)
Cell harvester (e.g. PerkinElmer)
Ultra-Turrax® (IKA, model: 0001602800 ) or similar disperser
Water bath
Scintillation counter
Software
Prism (Graphpad Software, San Diego, CA, USA) or similar
Procedure
Before the assay
Prepare assay and wash buffer.
With some receptor sources PEI-pretreated filterplates may improve the TB/SB ratio: Prepare 0.1% PEI solution and pipet 100 μl/filter on the filterplate, subsequently place in refrigerator (4 °C) for at least 2 hours.
The experiment is performed in 96 well plates (polysterene) with a total assay volume of 1,000 μl (450 μl assay buffer + 200 μl radioligand + 200 μl of assay buffer or 5 μM (+)-butaclamol + 150 μl membrane preparation).
Prepare NSB solution.
Prepare radioligand solutions.
Membrane preparation.
Prepare receptor-containing membrane suspension according to preparation protocol. Re-homogenize suspension in small volume (< 2 ml) using short burst of Ultra-Turrax. Dilute to desired protein concentration and to yield a total volume of about 4 ml per 24 data point experiment. The protein concentration of the membrane suspension should be chosen so that a robust specific binding signal is obtained but at the same time total binding should be < 10% (even better < 5%) of free radioligand concentration. Protein content can be assayed by a variety of essays, e.g. Bradford (1976). Prepare the membrane suspension initially in ice.
Pre warm all solutions for 15 min in 25 °C water bath.
Final preparation (Table 1), add components to wells in following order:
450 μl assay buffer.
200 μl assay buffer (TB) or 200 μl 5 μM butaclamol (NSB).
150 μl membrane suspension.
Start reaction by adding 200 μl radioligand.
Table 1. Pipetting scheme for sample on microtiter plate
T1
T1
T2
T2
T3
T3
T4
T4
T5
T5
T6
T6
TB1
TB1
TB2
TB2
TB3
TB3
TB4
TB4
TB5
TB5
TB6
TB6
NSB1
NSB1
NSB2
NSB2
NSB3
NSB3
NSB4
NSB4
NSB5
NSB5
NSB6
NSB6
During the assay
Incubate for 120 min at 25 °C in water bath.
Terminate reaction by rapid vacuum filtration over Whatman GF/C filters using a cell harvester. Wash filter 10 times with ice-cold wash buffer.
After the experiment
Add aliquots of 50 μl radioligand to wells of counting plate to determine total radioactivity.
Dry, e.g. in oven for 2 h.
Place sticker on bottom plate. Add scintillation cocktail (20 μl) to filters, place sticker on top of plate and count in a scintillation counter, allow adequate time (15 min) before counting samples.
Data analysis
The following calculations are required to derive Kd and Bmax from the data.
Carefully inspect raw data for consistency of replicates. Do not light-heartedly eliminate apparent ‘outliers’ from the data set.
Subtract mean non-specific binding from each replicate of total binding to obtain specific binding.
Transform totals to molar concentration of radioligand in the assay, and measured bound values (total binding, non-specific binding and specific binding) to molar amount of ligand based upon the specific radioactivity of the radioligand and the efficiency of the scintillation counter. A representative experiment with D2 receptors may look like this.
Plot specific binding (y-axis) vs. concentration of radioligand (x-axis).
Analyze data by non-linear iterative curve fitting using a rectangular hyperbolic function using one of many available iterative curve fitting programs, e.g. Prism.
Molar amount of Bmax can be corrected for tissue content, mostly the amount of protein per well (mostly yielding fmol/mg protein); alternative normalization e.g. for tissue wet weight, number of cells or DNA content are possible.
As internal quality check, determine the fraction of total binding from total radioactivity in the assay. This needs to be < 10% (better < 5%) as otherwise non-equilibrium conditions may exist. In the latter case, membrane concentration in the assay can be reduced if the measured signal remains robust. Alternatively, assay volume can be increased as this will increase total amount of radioligand (concentration constant) but not amount bound (membrane protein amount constant).
In the past when no computer assisted data analysis was available Kd and Bmax were estimated with the Rosenthal plots, better known as Scatchard plots (Rosenthal invented them but Scatchard made them famous). In these plots the specifically bound radioligand is on the x-axis and is plotted against the ratio of bound/unbound radioligand on the y-axis. The x-intercept corresponds to Bmax and the negative reciprocal of the slope corresponds to Kd. Rosenthal/Scatchard plots can still be useful to visualize that data points indeed fall on a linear line but the estimates derived from this are less reliable than those from iterative curve fitting (the assumptions of linear regression used in these plots don’t meet because x and y are not independent of each other), making this an outdated analysis.
If saturation binding experiments are performed in the absence and presence of an inhibitor, the data can be used to test for a competitive nature of the inhibitor. This is the case if the presence of inhibitor changes the Kd but not the Bmax of the radioligand.
Recipes
Assay buffer
50 mM TRIS: TRIS-HCl: 6.6 g and TRIS base 970 mg (or only 6.04 g TRIS base/L)
5 mM KCl: 373 mg/L
2 mM CaCl2: 220 mg/L
2 mM MgCl2(6 H2O): 410 mg/L
pH 7.4
Wash buffer
50 mM TRIS: TRIS-HCl (33 g/5 L) and TRIS base (4.85 g/5 L) (or only 30.22 g/5 L TRIS base)
pH 7.4
0.1% PEI (only for D3 receptor assay)
Prepare 0.1% solution, PEI is delivered as a 50% solution, pipet 1 ml from this with a syringe and add to 9 ml aqua dest. To get 5%, pipet 400 μl PEI 5% + 19.6 ml destilled water to get PEI 0.1%
NSB solution
3.98 mg butaclamol HCl/10 ml assay buffer yields 1.10-3 M (prepare aliquots of this)
Dilute this 1:200 to obtain 5 μM in solution, i.e. 1 μM final concentration in assay
Radioligand solutions
The intended radioligand concentration range in the assay should be chosen to cover both the expected Kd and about 5-10x Kd (Kd spiperone in transfected cells according to literature ≈ 0.05 nM, 0.35 nM and 0.07 nM for D2, D3 and D4, respectively). The stock solution to be prepared needs to be 5x the assay concentration. Thus, the stock solution should be 25-50x Kd. The following is an example calculation based on a specific activity of 16.2 Ci/mmol radioactive of the radioactive stock solution for a D2 receptor assay. This needs to be adapted for the other subtypes based on their Kd values and for each batch of radioligand and its specific activity.
[3H]-spiperone concentration = 1 mCi/ml/(16.2 Ci/mmol x 1,000 mCi/Ci) = 61.7 μM
Highest concentration needed (10 x Kd x 5) = 10 x 0.05 x 5 = 2.5 nM
2.5 ml x 2.5 nM = 0.00625 nmol [3H]-spiperone needed
(0.00625 nmol)/61.7 x 103 nM = 0.1013 μl of [3H]-spiperone solution needed for 2.5 ml
Thus 1 μl [3H]-spiperone solution in 25.0 ml assay buffer to yield a concentration of 2.5 nM.
A free tool for such calculations can be found at www.graphpad.com/quickcalcs/chemMenu/.
Radioligand Dilutions (Over a 100-fold Range) (Table 2).
Table 2. Dilution scheme to for radioligand working solutions
Number
Relative concentration
Preparation
1
100
2.5 ml of radioligand at the highest concentration
2
66.67
Mix 800 μl from tube 1 + 400 μl assay buffer
3
50
Mix 1,000 μl from tube 1 + 1,000 μl assay buffer
4
25
Mix 1,000 μl from tube 3 + 1,000 μl assay buffer
5
12.5
Mix 1,000 μl from tube 4 + 1,000 μl assay buffer
6
6.25
Mix 1,000 μl from tube 5 + 1,000 μl assay buffer
Acknowledgments
This protocol is the adaptation of a protocol originally published by Levant (2007).
References
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Levant, B. (2007). Characterization of Dopamine Receptors. Curr Protoc Pharmacol 36:1.6.1–1.6.15.
van Wieringen, J. P., Booij, J., Shalgunov, V., Elsinga, P. and Michel, M. C. (2013). Agonist high- and low-affinity states of dopamine D2 receptors: methods of detection and clinical implications. Naunyn Schmiedebergs Arch Pharmacol 386(2): 135-154.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Wieringen, J. V. and Michel, M. C. (2013). [3H]-Spiperone Saturation Binding to Dopamine D2, D3 and D4 Receptors. Bio-protocol 3(20): e945. DOI: 10.21769/BioProtoc.945.
Download Citation in RIS Format
Category
Neuroscience > Sensory and motor systems
Biochemistry > Protein > Interaction > Protein-ligand interaction
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946 | https://bio-protocol.org/en/bpdetail?id=946&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Trehalase Activity in Arabidopsis thaliana Optimized for 96-well Plates
Hilde Van Houtte
Patrick Van Dijck
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.946 Views: 11851
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Plant Physiology Mar 2013
Abstract
Trehalose is a nonreducing disaccharide. It is a common sugar in bacteria, fungi and yeast, where it functions as a carbon source and stress protectant. In contrast, plants, although encoding large trehalose biosynthesis gene families, contain only small amounts of trehalose. The intermediate compound of trehalose, trehalose-6-phosphate (T6P), is a signaling molecule in plants, regulating metabolism, growth, and development. Most plants contain only a single trehalase, the enzyme that specifically hydrolyzes trehalose into two glucose molecules. High trehalase activity has been suggested to be part of the defense mechanism in plants hosting mycorrhizal fungi, rhizobia, and the plant pathogen Plasmodiophora brassica. Recently, it was shown in Arabidopsis thaliana that high trehalase activity is associated with an increase in drought stress tolerance and that trehalase fulfills an important role in stomatal regulation. Here we describe a protocol for measuring trehalase activity in Arabidopsis tissues, optimized for 96-well plates. Dialyzed protein extracts will be incubated with trehalose, followed by the quantitation of the released glucose using glucose oxidase-peroxidase.
Keywords: Arabidopsis thaliana Trehalose Trehalase
Materials and Reagents
Plant tissues
Liquid nitrogen
MES
Phenylmethylsulfonyl fluoride (PMSF)
EDTA
Polyvinylpyrrolidone (PVP)
Dithiothreitol (DTT)
CaCl2
Glucose
Bovine serum albumin (BSA)
Na2CO3
K-Na-tartrate
CuSO4.5H2O
KOH
NaOH
Folin & Ciocalteu's phenol reagent (Sigma-Aldrich, catalog number: 47641 )
Trehalose (Sigma-Aldrich, catalog number: T9531 )
Glucose, GOD-PAP (DIALAB GmbH, catalog number: D95218B )
Extraction buffer (see Recipes)
Dialysis buffer (see Recipes)
Trehalose buffer (see Recipes)
Glucose standards (see Recipes)
BSA standards (see Recipes)
Lowry buffers (see Recipes)
Equipment
Mortars and pestles
Spectra/Por® 1 Dialysis Membrane (IEEE, catalog number: 132660 )
Note: 96-well dialysis systems can be used for the dialysis of multiple samples.
Transparent 96-well plates with flat bottom
Rocker
Refrigerated microcentrifuge
Pre-chilled microcentrifuge tubes
100 ml cylinder
Magnetic stir plate and magnet
Cold room
Spectrophotometric plate reader
Procedure
Protein extraction
Note: Work always on ice unless stated differently.
Grind plant tissues in liquid nitrogen with mortar and pestle.
Aliquot 100 mg tissue powder in chilled microcentrifuge tubes. Prepare at least 3 replicates per sample.
Add 1 ml of ice cold extraction buffer to each sample. Homogenize samples by pipetting up and down. Centrifuge at 18,000 x g, 4 °C, 10 min.
Transfer the supernatant to a new, chilled microcentrifuge tube. The obtained protein extracts can be stored at -80 °C.
Dialysis
Wet a piece of Spectra/Por?1 Dialysis Membrane with water and tie a knot at the bottom (Figure 1).
Transfer 500 μl of the protein extract into the tubing and tie a knot at the top (Figure 1).
Figure 1. Tying a knot at the bottom and top of a dialysis membrane
Place tubing in a 100 ml cylinder filled with ice cold dialysis buffer. Dialyze the extract at 4 °C for 2-3 h under continuous stirring on a magnetic stir plate.
Replace dialysis buffer and continue the dialysis at 4 °C overnight.
Transfer the dialyzed extracts to new, chilled microcentrifuge tubes. The dialyzed extracts can be stored at -80 °C.
Trehalase activity adapted for 96-well plates
Note: Work always on ice unless stated differently.
Prepare a water bath at 95 °C.
For samples
Transfer 10 μl of the dialysis product in a 96-well plate with flat bottom (= plate S).
Transfer 10 μl of each respective glucose standard to plate S.
For blanks
Transfer 10 μl of the dialysis product in a 96-well plate with flat bottom (= plate B).
Place plate B at 95 °C temperature for 5 minutes to denature the trehalase enzyme present in the blanks.
Place plate B on ice for 2 min.
For samples and blanks
Add 50 μl of trehalose buffer to plates S and B. Mix by pipetting up and down.
Incubate plates S and B for 30 min on a rocker at 30 °C.
Stop the reaction by boiling for 5 min at 95 °C (denaturation of the trehalase).
Place plates S and B on ice for 2 min.
Add 200 μl of Glucose, GOD-PAP to plates S and B for determining the glucose concentration of the samples and blanks by colorimetry. Mix by pipetting up and down.
Incubate plates S and B for 15 min at 30 °C on a rocker.
Measure the absorbance of plates S and B at 505 nm with a spectrophotometric plate reader (30 °C).
Determine the glucose standard curve to calculate the glucose concentration (nmol) present in the samples and blanks. Subtract the glucose concentration of the blanks from the samples (see Example: Calculating the trehalase activity in excel).
For proteins (Lowry procedure, Van Houtte et al., 2013)
Transfer 10 μl of the dialysis product in a 96-well plate with flat bottom (= plate P) and add 30 μl water to these samples.
Transfer 40 μl of each respective BSA standard to plate P.
Add 200 μl of Reagent C (Lowry buffers) to plate P and incubate for 10 min at room temperature.
Add 20 μl of Reagent D (Lowry buffers) to plate P and incubate for 30 min at 30 °C.
Measure the absorbance of plate P at 546 nm with a spectrophotometric plate reader (30 °C).
Use the BSA standard curve to determine the amount of protein (μg) present in the extracts (see Example: Calculating the trehalase activity in excel).
Express the trehalase specific activity as nmol of glucose released per min per mg protein (see Example: Calculating the trehalase activity in excel).
Data analysis
Here we show an example how to calculate the trehalase activity from a protein extract of Arabidopsis Col-0 seedlings.
Glucose standard curve
Using excel, plot the glucose concentration of the respective glucose standards on the X axis and the corresponding absorbances (505 nm) on the Y axis (Table 1; Figure 2). Add a linear trendline to the glucose standard curve and diplay its equation (Figure 2; [1]).
[1] y = 0.0157x + 0.0858
Table 1. Absorbances at 505 nm (Plates S and B)
Glucose standards
Absorbance (505 nm)
0 nmol glucose
0.08
20 nmol glucose
0.3721
40 nmol glucose
0.7421
60 nmol glucose
1.0355
80 nmol glucose
1.3792
100 nmol glucose
1.6143
Arabidopsis Col-0 tissues
Absorbance (505 nm)
Sample
0.1483
Blank
0.0898
Figure 2. Glucose standard curve
Determination of the glucose concentration
Equation [1] and the absorbances (505 nm) of the sample and blank (Table 1) can be used to calculate the glucose concentration present in the sample and blank.
[2] Nmol glucose in sample = (0.1483-0.0858)/0.0157
= 3.9809
[3] Nmol glucose in blank = (0.0898-0.0858)/0.0157
= 0.2548
In order to know how many glucose is released per min in the extract, subtract [3] from [2], and divide by the duration of the incubation time (min).
[4] Nmol glucose released per min = (3.9809 - 0.2548)/30
= 0.1242
BSA standard curve
Since trehalase activity is expressed per unit of protein, we need to determine the amount of protein present in the extract. Using excel, plot the protein content of the respective BSA standards on the X axis and the corresponding absorbances (546 nm) on the Y axis (Table 2; Figure 3). Add a linear trendline to the BSA standard curve and display its equation (Figure 3; [5]).
Figure 3. BSA standard curve
[5] y = 0.0242x + 0.0576
Determination of the protein content
Equation [5] and the absorbance (546 nm) of the protein extract (Table 2) can be used to calculate the protein content.
Table 2. Absorbances at 546 nm (Plate P)
BSA standards
Absorbance (546 nm)
0 μg protein
0.0488
10 μg protein
0.3224
20 μg protein
0.5603
30 μg protein
0.7159
40 μg protein
1.0637
Arabidopsis Col-0 tissues Absorbance (505 nm)
Protein 0.4786
[6] μg protein in extract = (0.4786 - 0.0576)/0.0242
= 17.3967
Trehalase specific activity of Arabidopsis Col-0 seedlings
Since the specific trehalase activity is expressed as nmol glucose produced per min per mg protein, we need to divide [4] by [6], and multiply by 1,000.
[7] Trehalase specific activity in nmol glucose per min per mg protein = 0.1242/17.3967*1,000
= 7.1393
Recipes
Extraction buffer
0.1 M MES-KOH, pH 6
1 mM PMSF
1 mM EDTA
1% (w/v) PVP
1 mM DTT
Stored at 4 °C
Dialysis buffer
10 mM MES-KOH, pH 7
50 μM CaCl2
Stored at 4 °C
Trehalose buffer
250 mM trehalose
62.5 mM MES-KOH, pH 7
125 μM CaCl2
Stored at 4 °C
Glucose standards
Make standards with 10, 8, 6, 4, 2 and 0 μl of a 10 mM glucose solution in a total V of 10 μl
Fresh prepared or store 10 μl aliquots at -20 °C
BSA standards
Make standards with 40, 30, 20, 10 and 0 μl of a 1 mg/ml BSA solution in a total V of 40 μl
Fresh prepared and keep on ice
Lowry buffers
Reagent A:
2% (w/v) Na2CO3, 0.02% (w/v) K-Na-tartrate in 0.1 M NaOH
Stored at room temperature
Reagent B:
1% (w/v) CuSO4.5H2O
Stored at room temperature
Reagent C:
Mix solution A and B (100:1, [v/v])
Fresh prepared
Reagent D:
Mix Folin & Ciocalteu's phenol reagent with MilliQ water (1:2, [v/v])
Fresh prepared
Acknowledgments
This protocol was developed in the framework of the following paper: Van Houtte et al. (2013). It was developed based on two previous publications: Brodmann et al. (2002) and Pernambuco et al. (1996). Hilde Van Houtte was supported by the KU Leuven industrial research fund (IOF/KP/08/001). This work was supported by a grant from the FWO (G.0859.10).
References
Brodmann, A., Schuller, A., Ludwig-Muller, J., Aeschbacher, R. A., Wiemken, A., Boller, T. and Wingler, A. (2002). Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae. Mol Plant Microbe Interact 15(7): 693-700.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent.J Biol Chem 193(1): 265-275.
Pernambuco, M. B., Winderickx, J., Crauwels, M., Griffioen, G., Mager, W. H. and Thevelein, J. M. (1996). Glucose-triggered signaling in Saccharomyces cerevisiae: different requirements for sugar phosphorylation between cells grown on glucose and those grown on non-fermentable carbon sources. Microbiology 142(7): 1775-1782.
Van Houtte, H., Vandesteene, L., Lopez-Galvis, L., Lemmens, L., Kissel, E., Carpentier, S., Feil, R., Avonce, N., Beeckman, T., Lunn, J. E. and Van Dijck, P. (2013). Overexpression of the trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in abscisic acid-induced stomatal closure. Plant Physiol 161(3): 1158-1171.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Houtte, H. V. and Dijck, P. V. (2013). Trehalase Activity in Arabidopsis thaliana Optimized for 96-well Plates. Bio-protocol 3(20): e946. DOI: 10.21769/BioProtoc.946.
Van Houtte, H., Vandesteene, L., Lopez-Galvis, L., Lemmens, L., Kissel, E., Carpentier, S., Feil, R., Avonce, N., Beeckman, T., Lunn, J. E. and Van Dijck, P. (2013). Overexpression of the trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in abscisic acid-induced stomatal closure. Plant Physiol 161(3): 1158-1171.
Download Citation in RIS Format
Category
Plant Science > Plant biochemistry > Protein > Activity
Biochemistry > Protein > Isolation and purification
Biochemistry > Carbohydrate > Disaccharide
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947 | https://bio-protocol.org/en/bpdetail?id=947&type=0 | # Bio-Protocol Content
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Peer-reviewed
Soft Agar Anchorage-independent Assay
LW Li-Ting Wang
SH Shih-Hsien Hsu
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.947 Views: 14533
Reviewed by: Lin FangPinchas Tsukerman Fanglian He
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Original Research Article:
The authors used this protocol in Cancer Research Jan 2013
Abstract
Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, soft agar anchorage-independent assay were used to determine tumor transform activity of hepatoma cells with ISX over expression or knockdown in vitro.
Materials and Reagents
HCC cells (Hep G2: ATCC® HB-8065TM and Hep 3B: ATCC® HB-8064TM)
ISX fusion GFP expression plasmid or ISX shRNAi
Agarose-LE (MDBio)
2x MEM no phenol red (Gibco)
100x NEAA (Gibco)
FBS (Gibco)
10x PBS (MDBio)
Crystal violet (Sigma-Aldrich, catalog number: C3866 )
Methanol
Ethanol
ddH2O
Equipment
6 well culture dishes (Greiner Bio-One GmbH)
Water bath
Cell counter
37 °C incubator
Procedure
HCC cells transfected with ISX fusion GFP expression plasmid or ISX shRNAi and then were selected to stable clones.
The stable HCC clones were then grown in MEM culture medium supplemented with 10% FBS and 1x NEAA according to ATCC guidelines.
Prepare 0.6% and 1.2% agar in ddH2O by autoclave and keep warm in 65 °C water bath.
Making bottom agar: adding 1 ml of 2x MEM culture medium with 20% FBS to 1 ml 1.2% agar. After well mixing, the mixture was put into one well of six-well culture dish to form bottom gel layer.
The stable HCC cells were harvested and counting the cell numbers. Dilute the cells to 1 x 104 cells per ml in 2x MEM with 20% FBS. Adding 1 ml diluted HCC stable cells into 1 ml 0.6% agar. After well mixing, the mixture were put on the top of bottom agar per well. The cell-agar mixture became solid phase at 37 °C incubator for 30 minutes. Then, 2 ml 10% FBS MEM culture medium were added on the top agar in each well.
These dishes were then cultured at 37 °C incubator for 2 weeks and changed the culture medium for each 3 days.
Colonies were visualized by staining with 0.05% crystal violet-75% ethanol or 40% Methanol to 0.45 μm filter. Colonies larger than 0.5 mm were counted.
Acknowledgments
This protocol was adapted from Hsu et al. (2013).
References
Hsu, S. H., Wang, L. T., Lee, K. T., Chen, Y. L., Liu, K. Y., Suen, J. L., Chai, C. Y. and Wang, S. N. (2013). Proinflammatory homeobox gene, ISX, regulates tumor growth and survival in hepatocellular carcinoma. Cancer Res 73(2): 508-518.
Article Information
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© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Wang, L. and Hsu, S. (2013). Soft Agar Anchorage-independent Assay. Bio-protocol 3(20): e947. DOI: 10.21769/BioProtoc.947.
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Category
Cancer Biology > General technique > Cell biology assays > Cell viability
Cell Biology > Cell staining > Whole cell
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948 | https://bio-protocol.org/en/bpdetail?id=948&type=0 | # Bio-Protocol Content
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Peer-reviewed
Xenograft Tumor Growth Assay
LW Li-Ting Wang
SH Shih-Hsien Hsu
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.948 Views: 13306
Reviewed by: Lin FangPinchas Tsukerman Fanglian He
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Original Research Article:
The authors used this protocol in Cancer Research Jan 2013
Abstract
Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, Xenograft tumor assay was used to determine the tumorigenic activity of hepatoma cells with ISX over expression on nude mice in vivo.
Materials and Reagents
HCC cells (Hep G2: ATCC® HB-8065 TM and Hep 3B: ATCC® HB-8064 TM)
CAnN.Cg-Foxn1nu/CrlBltw Nude mice
ISX fused with GFP or ISX shRNAi expression constructs
PBS (MDBio)
2.5% Trypsin (10x) (Gibco , catalog number: 15090-046 )
MEM Culture medium (Gibco, catalog number: 61100-061 )
Equipment
100 mm2 plate
15 ml conical tubes
Centrifuge (Beckman Coulter)
Eppendorf
1 ml syringes (0.45 x 13 mm)
Tissue culture hood
Caliper (Digital Caliper)
Procedure
HCC cells were transfected with ISX fused with GFP or ISX shRNAi expression constructs and selected into stable clones.
Determining the cells number for injection. 5 x 107 cells per ml will be required to trypsinizing (usually a 100% confluent plate of 100 mm2 will yield at least 6 injections at 5 x 106 cells/injection).
Trypsinizing the cells with 1x trypsin solution from needed number of plates to be counted all at once.
Collecting the detached cells in 15 ml conical tubes and spin for 3 min at 300 x g.
To remove the supernatant and re-suspend the cells with 3 ml of culture medium for counting.
To remove three 10 μl aliquots into 3 separate eppendorfs and dilute each 10 μl 1:100 by adding 990 μl of culture medium, mix well for counting.
Then, to remove 10 μl of 1:10 dilutions for counting, counting each of three dilutions and average the three numbers.
Determining the concentration of cells in cells/ml by using the following formula: Average counts x 10,000 x dilution factor (1,000) = #cells/ml.
Determining the volume required to add to achieve final concentration of cells for injection per volume to be injected (i.e. 5 x 106 cells/ml injections).
Spin down 15 ml conical for 3 min at 300 x g.
Discard supernatant and re-suspend the pellet in the previously determined volume from step 8.
Draw up each injection/mouse in 1 ml syringes in the tissue culture hood prior to going to the animal facility. Place the separate syringes each containing 200 μl on ice.
The tumor volume was estimated according to the formula: volume (cm3) = 1/2(L x W2), where L and W are the length and width of the tumor, respectively.
Acknowledgments
This protocol was adapted from Hsu et al. (2013).
References
Hsu, S. H., Wang, L. T., Lee, K. T., Chen, Y. L., Liu, K. Y., Suen, J. L., Chai, C. Y. and Wang, S. N. (2013). Proinflammatory homeobox gene, ISX, regulates tumor growth and survival in hepatocellular carcinoma. Cancer Res 73(2): 508-518.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Wang, L. and Hsu, S. (2013). Xenograft Tumor Growth Assay. Bio-protocol 3(20): e948. DOI: 10.21769/BioProtoc.948.
Download Citation in RIS Format
Category
Cancer Biology > Proliferative signaling > Animal models > Proliferation analysis
Cancer Biology > Inflammation > Tumor formation > Cell invasion
Biochemistry > Protein > Expression
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949 | https://bio-protocol.org/en/bpdetail?id=949&type=0 | # Bio-Protocol Content
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Peer-reviewed
Cell Fractionation and Quantitative Analysis of HIV-1 Reverse Transcription in Target Cells
Vaibhav B Shah
CA Christopher Aiken
Published: Vol 3, Iss 20, Oct 20, 2013
DOI: 10.21769/BioProtoc.949 Views: 10010
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Original Research Article:
The authors used this protocol in Journal of Virology Jan 2013
Abstract
This is a protocol to detect HIV-1 reverse transcription products in cytoplasmic and nuclear fractions of cells infected with VSV-G-pseudotyped envelope-defective HIV-1. This protocol can also be extended to HIV-1 with regular envelope.
Keywords: HIV-1 Reverse Transcription Cytoplasm Nucleus Quantitative PCR
Materials and Reagents
HEK 293T cells
HeLa cells
Dulbecco’s Modified Eagle Medium (DMEM) (Mediatech, Cellgro®, catalog number: 10-013-CV )
R9-ΔE plasmid ((Zhou and Aiken, 2001), an HIV-1 proviral DNA clone created by introducing a frameshift mutation in envelope of the wild-type infectious R9 clone. Virions produced by this clone are non-infectious but can be made infectious by pseudotyping with envelopes from VSV or other viruses)
pHCMV-G (VSV-G) plasmid ((Yee et al., 1994), a retrovirus-derived plasmid in which the retroviral envelope glycoprotein is replaced with glycoprotein from vesicular stomatitis virus [VSV]).
p24 ELISA kit (in-house)
Phosphate-buffered saline (PBS) (Mediatech, Cellgro®, catalog number: 21-0310-CV )
VSV-G-pseudotyped envelope-defective HIV-1 (R9-ΔE) virus particles
Efavirenz (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH, catalog number: 11680 )
DNase I (Roche, catalog number: 10104159001 )
0.25% Trypsin/2.21 mM EDTA (Mediatech, Cellgro®, catalog number: 25-053-CI )
Triton X-100 (Mallinckrodt, catalog number: 9002-93-1 )
DNeasy blood and tissue kit (QIAGEN, catalog number: 69506 )
cOmplete, Mini, EDTA-free protease-inhibitor cocktail tablet (Roche, catalog number: 11836170001 )
4 to 20% Polyacrylamide gradient Tris-glycine gels (Bio-Rad)
Nitrocellulose membrane (General Electric Company)
Mouse monoclonal anti-GAPDH antibody (Santa Cruz, catalog number: sc-47724 )
Mouse monoclonal anti-LaminB1 antibody (Life Technologies, catalog number: 33-2000 )
SYBR green (ABI, catalog number : 4309155 )
DpnI (New England Biolabs, catalog number: R0176L )
DTT
Yeast tRNA (Roche, catalog number : 10109541001 )
Forward primer MH531 (5’-TGTGTGCCCGTCTGTTGTGT-3’)
Reverse primer MH532 (5’-GAGTCCTGCGTCGAGAGAGC-3’)
DNase/RNase-free water
SDS-PAGE sample buffer
Sodium deoxycholate (Sigma-Aldrich, catalog number: 30970 )
N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES) (Sigma-Aldrich, catalog number: B4554 )
Hypotonic buffer (see Recipes)
Radioimmunoprecipitation buffer (see Recipes)
2x BES-buffered saline (BBS) (see Recipes)
Equipment
10 cm cell culture dish
0.45-μm-pore-size syringe filters (Thermo Fisher Scientific, catalog number: 190-2545 )
0.20- μm-pore-size syringe filters (Thermo Fisher Scientific, catalog number: 190-2520 )
1.5 ml screw-cap tube
Tabletop centrifuge (Thermo Fisher Scientific, Sorvall®)
Tabletop refrigerated centrifuge (Thermo Fisher Scientific)
Mx-3000p thermocycler (Stratagene)
CO2 incubator
Procedure
Production of VSV-G-pseudotyped envelope-defective HIV-1 (R9-ΔE clone) virus particles (Aiken, 1998)
Culture 293T cells in DMEM containing 10% v/v fetal bovine serum (FBS) and supplemented with antibiotics [Penicillin (100 IU/ml) and Streptomycin (100 μg/ml)] at 37 °C, 5% CO2.
Detach cells from a nearly confluent culture dish with the help of 0.25% Trypsin/2.21 mM EDTA and seed 2 x 106 cells in 9 ml medium per 100 mm culture dish and incubate at 37 °C.
Transfect of 293T cells next day using the calcium phosphate-BBS method (Chen and Okoyama, 1987).
Mix 15 μg of R9-ΔE and 5 μg of pHCMV-G (VSV-G) plasmids in a tube.
Add 0.2 μm filtered water to the tube to make up the volume to 450 μl.
Add 50 μl of 2.5 M CaCl2 to the tube.
Add 500 μl of 2x BBS to the tube dropwise.
Gently mix the contents of the tube by pipetting few times.
Incubate the tube at room temperature for 20 to 30 min.
Add the mixture to 293T cells with gentle swirling and incubate cells at 35 °C and 3% CO2.
Aspirate media from the transfected dish ~16 h after transfection, wash cells with 5 ml PBS, replenish with 5 ml of fresh cell culture media and incubate at 37 °C, 5% CO2.
Two days after transfection, harvest culture supernatant containing virus particles, centrifuge at 1,500 x g for 5 min to pellet cells and debris.
Filter the supernatant through 0.45-μm-pore-size syringe filters, aliquot and freeze at -80 °C.
Infection of HeLa cells with VSV-G-pseudotyped envelope-defective HIV-1 (R9-ΔE)
Plate HeLa cells at a density of 1.5 x 105 cells/well in 12-well plates (1 ml total culture volume per well).
24 hours later treat virus inocula with DNase I (20 μg/ml) plus MgCl2 (10 mM) and incubate in a water bath at 37 °C for 1 h.
Infect cells with DNase I-treated inocula equivalent to 15 ng of p24 (determined by p24 ELISA using in-house kit (Wehrly and Chesebro, 1997)).
Perform parallel infection in the presence of efavirenz (1 μM) to define the residual plasmid DNA levels carried over from transfection.
Incubate infected cells at 37 °C for 8 h.
Note: One can also analyse time course of reverse transcription by harvesting infected cells at different time intervals after infection.
Cell fractionation of HIV-1 infected HeLa cells
After incubation for desired time, aspirate culture media and wash cells once with PBS.
Dislodge adherent cells by incubation with 500 μl of 0.25% Trypsin-EDTA at 37 °C for 2 min.
Collect trypsinized cells in a 1.5 ml screw-cap tube. Centrifuge at 300 x g for 5 min to pellet cells.
Lyse cell pellets in 200 μl of hypotonic buffer containing 0.1% Triton-X-100 and incubate on ice for 15 min.
Note: Concentration of Triton-X-100 was optimized for HeLa cells. The concentration of Trition X-100 represents the lowest concentration at which about 95% of the cells counted under the microscope had intact nuclei but no plasma membrane.
Centrifuge at 17,000 x g for 5 min at 4 °C and collect the supernatant as cytoplasmic fraction.
Wash the nuclear pellet with 1 ml hypotonic buffer without Triton-X-100 thrice. After each wash centrifuge at 17,000 x g for 5 min at 4 °C to pellet the nuclei and aspirate off supernatant.
Isolate DNA from nuclear pellet using DNeasy blood and tissue kit as per manufacturer’s protocol. Elute DNA in the last step in a fresh collection tube using 100 μl DNase/RNase-free water. Eluted DNA can be stored at -80 °C or used directly to perform qPCR.
In parallel, prepare whole-cell, cytoplasmic and nuclear lysates from uninfected cells to check for cytoplasmic contamination of nuclear fractions.
To prepare whole cell lysate, lyse cells in radioimmunoprecipitation (RIPA) buffer (follow steps C2-C5 except the use of RIPA buffer instead of hypotonic buffer). Add equal volume of 2x SDS-PAGE sample buffer for gel electrophoresis and heat at 95 °C in a heat block for 5 min.
Prepare cytoplasmic lysate as described above (steps C2 to C5). Add equal volume of 2x SDS-PAGE sample buffer for gel electrophoresis and heat at 95 °C in a heat block for 5 min.
To prepare nuclear lysate, follow steps C2 to C6, and then lyse the nuclear pellet in 1x SDS-PAGE sample buffer. Heat at 95 °C in a heat block for 5 min. Resolve equal volumes of whole cell, cytoplasmic and nuclear lysates on a 4-20% polyacrylamide gradient Tris-glycine gel.
Transfer resolved proteins onto a nitrocellulose membrane.
Block the membrane with 5% non-fat milk solution in PBS and probe with anti-GAPDH and anti-LaminB1 antibodies (concentrations recommended by manufacturer) followed by appropriate secondary antibodies (concentrations recommended by manufacturer) as cytoplasmic and nuclear markers respectively.
SYBR green-based Quantitative PCR for quantitation of viral reverse transcription products
Treat isolated DNA from step C7 with DpnI (17 μl DNA + 2 μl buffer + 1 μl of DpnI-20 units) by incubation at 37 °C for 1 to 2 h. Inactivate DpnI by incubation at 80 °C for 20 min.
Quantitation of viral reverse transcription products.
Prepare reaction mixture by mixing DNA (5 μl), PCR mix containing SYBR green (12.5 μl), forward primer (150 nM), reverse primer (150 nM) and tRNA (1 μg/μl) containing DNase/RNase-free water up to 25 μl.
Prepare standards ranging from 10 to 109 copies/reaction of R9-ΔE plasmid. Dilutions of standards should be made in 1 μg/μl tRNA-containing water.
Set PCR reaction using the following thermal profile:
Recipes
Hypotonic buffer
10 mM Tris pH 8.0
10 mM KCl
1.5 mM MgCl2
1 mM DTT
Protease inhibitor cocktail (one tablet per 10 ml of buffer)
Radioimmunoprecipitation buffer
50 mM Tris pH 7.5
1% Triton-X-100
250 mM NaCl
5 mM EDTA
0.1% SDS
1% sodium deoxycholate
Protease inhibitors cocktail (one tablet per 10 ml of buffer)
2x BES-buffered saline (BBS)
50 mM BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid)
1.5 mM Na2HPO4
280 mM NaCl
pH 6.95
Acknowledgments
This protocol is adapted from Shah et al (2013). This protocol was supported by NIH grant AI076121 to C.A.
References
Aiken, C. (1998). Mechanistic independence of Nef and cyclophilin A enhancement of human immunodeficiency virus type 1 infectivity. Virology 248(1): 139-147.
Chen, C. and Okayama, H. (1987). High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7(8): 2745-2752.
Shah, V. B., Shi, J., Hout, D. R., Oztop, I., Krishnan, L., Ahn, J., Shotwell, M. S., Engelman, A. and Aiken, C. (2013). The host proteins transportin SR2/TNPO3 and cyclophilin A exert opposing effects on HIV-1 uncoating. J Virol 87(1): 422-432.
Wehrly, K. and Chesebro, B. (1997). p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents. Methods 12(4): 288-293.
Yee, J. K., Friedmann, T. and Burns, J. C. (1994). Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol 43 Pt A: 99-112.
Zhou, J. and Aiken, C. (2001). Nef enhances human immunodeficiency virus type 1 infectivity resulting from intervirion fusion: evidence supporting a role for Nef at the virion envelope. J Virol 75(13): 5851-5859.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Shah, V. B. and Aiken, C. (2013). Cell Fractionation and Quantitative Analysis of HIV-1 Reverse Transcription in Target Cells. Bio-protocol 3(20): e949. DOI: 10.21769/BioProtoc.949.
Download Citation in RIS Format
Category
Microbiology > Microbial genetics > RNA > qRT-PCR
Microbiology > Microbe-host interactions > Virus
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95 | https://bio-protocol.org/en/bpdetail?id=95&type=1 | # Bio-Protocol Content
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This is an In Press version of the protocol that has not yet been assigned to an issue.
Peer-reviewed
Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium
Xiyan Li
In Press
Published: Jul 20, 2011
DOI: 10.21769/BioProtoc.95 Views: 65130
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Abstract
Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, are used as medium to introduce the target gene expression cassette into benthamiana mesophyll cells.
Keywords: Transient expression Tobacco Agrobacterium Fluorescence Leaves
Materials and Reagents
Agrobacterium strain hosting a plant expression construct (usually driven by Cauliflower mosaic virus 35S promoter)
Healthy Nicotiana benthamiana (N. benthamiana) plants 2-4 weeks
MES
MgCl2 stock
Antibiotics
Acetosyringone
LB media with appropriate antibiotics (see Recipes)
Acetosyringone stock (see Recipes)
MES-K (see Recipes)
Resuspension solution (see Recipes)
Acetosyringone datasheet (Sigma-Aldrich) (see Recipes)
Equipment
Centrifuge for 50 ml tubes
Spectrometer
Syringe
UV lamp (optional)
Fluorescence microscope (optional)
Confocal laser scanning microscope (optional)
Procedure
Inoculate one single colony of Agrobacterium in 5 ml LB with appropriate antibiotics. Grow overnight at 28-30 °C.
Note: I usually use 100 μg/ml gentamicin (maintain the virulence of Agrobacterium strain GV3101) and 50 μg/ml spectinomycin (selective marker for shuttle vector) for most of the shuttle vectors.
Use 1 ml of the overnight culture to inoculate 25 ml LB (with same antibiotics, plus 20 μM acetosyringone added after autoclaving and immediately before use) and grow overnight.
Measure the A600 of overnight culture.
Precipitate the bacteria (5,000 x g, 15 min), resuspend the pellet in Resuspension Solution. The final A600 should be adjusted to 0.4.
Leave on the bench (room temperature) for 2-3 h (or overnight) before infiltration.
Perform the infiltration with 5 ml syringe. Simple press the syringe (no needle) on the underside of the leaf (Note: Avoid cotyledons), and exert a counter-pressure with finger on the other side. Successful infiltration is often observed as a spreading “wetting” area in the leaf.
(Optional) Check the GFP fluorescence by a portable long-wavelength UV lamp 2-5 days after infiltration. This only applies to strong expression of GFP signal (as green from red background).
Observe the fluorescence labeled protein under a fluorescent microscope or confocal laser scanning microscope. Or harvest leaves for protein purification.
Recipes
LB media with appropriate antibiotics
Usually two antibiotics used: one to maintain Agrobacteria virulence, one for the shuttle vector
Acetosyringone stock
100 mM in ethanol, stored at -20 °C
MES-K (0.5 M) (pH 5.6)
First make 0.5 M MES, adjust pH with KOH to 5.6
Resuspension solution
10 mM MgCl2
10 mM MES-K (pH 5.6)
Autocloave 15 min
100 μM acetosyringone (note: Added after autoclaving and immediately before using)
Acetosyringone datasheet
Synonyms 3’, 5’-Dimethoxy-4’-hydroxyacetophenone
Synonyms Acetosyringone
4’-Hydroxy-3’, 5’-dimethoxyacetophenone
Molecular Formula C10H12O4
Molecular Weight 196.20
CAS Number 2478-38-8
Beilstein Registry Number 1966119
EG/EC Number 2196105
MDL number MFCD00008748
References
Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F. and Sze, H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiol 147(4): 1675-1689.
Article Information
Copyright
© 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Plant Science > Plant transformation > Agrobacterium
Biochemistry > Protein > Expression
Molecular Biology > Protein > Expression
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How many plants are required for studying transient expression of a single gene?
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950 | https://bio-protocol.org/en/bpdetail?id=950&type=0 | # Bio-Protocol Content
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Peer-reviewed
Quantification of Bacterial Fatty Acids by Extraction and Methylation
MP Mark Politz
RL Rebecca Lennen
BP Brian Pfleger
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.950 Views: 14759
Reviewed by: Fanglian HeLin Fang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Journal of Bacteriology Jan 2013
Abstract
This protocol describes two similar methods for the extraction and methylation of fatty acids from bacterial cultures. The acid derivatization protocol (Lennen et al., 2013; Bligh and Dyer, 1959) results in the extraction and methylation of all fatty acids, both free and bound, from a bacterial culture, while the base derivatization protocol (Lennen and Pfleger, 2013) captures only bound (phospholipid, acyl-thioester) species. After extraction into hexane, the lipids may be analyzed by gas chromatography.
Keywords: Free Fatty Acid Methylation Fatty Acid Methyl Ester
Materials and Reagents
Cell culture for lipid analysis. When analyzing bacterial cultures expressing a thioesterase, it is advisable to wait until the late stationary phase (8-24 h for E. coli) to perform the extraction to allow sufficient time for product accumulation (Lennen et al., 2013)
Methyl heptadecanoate (should be ≥ 99% purity) (e.g. Sigma-Aldrich, catalog number: 51633 )
GC sample vials (VWR International, catalog number: 46610-722 ) (ensure you purchase vials compatible with any autosampler you are using)
Compressed nitrogen gas/regulator/tubing
Reverse osmosis water
Methanol
Absolute (100%) ethanol
High resolution gas chromatography grade hexane (99.9% pure mixture of hexane isomers)
Glacial acetic acid (Thermo Fisher Scientific, catalog number: A38-212 )
Deionized water
Anhydrous 1.25 M HCl in methanol (Sigma-Aldrich, catalog number: 17935 )
Sodium bicarbonate
0.5 M Sodium methoxide in methanol (Sigma-Aldrich, catalog number: 403067 )
Chloroform
Appropriate internal standards (e.g. non-native, odd chain fatty acids). Some possibilities include:
Heptadecanoic acid (C17:0) (99% + pure)
Pentadecanoic acid (C15:0) (99% + pure)
1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine in chloroform (99% + pure) (Avanti Polar Lipids, catalog number: 850704 )
External standards (the methyl esters of fatty acids you wish to quantify at a known concentration generally these may be obtained from a commercial source). A useful external standard mixture is sold by Supelco through Sigma-Aldrich (catalog number: 18918-1AMP )
Antifoam 204 (Sigma-Aldrich, catalog number: A8311 )
Equipment
5 ml glass pipettes
10 ml Glass centrifuge tubes (Thermo Fisher Scientific, KimbleTM, catalog number: 73785-10 ) with fluroropolymer lined caps (Thermo Fisher Scientific, KimbleTM, catalog number: 73802-15415 )
Gloves
Goggles
Appropriate personal protective equipment according to local regulations
Chemical fume hood
Centrifuge
Vortex mixer
Vacuum source/aspiration equipment
Lyophilizer
Water bath
Mass spectrometer or flame ionization detector
Agilent 7890 GC with a 30 m x 0.25 mm HP 5-ms capillary column
Procedure
Collect 2.5 ml of cell culture in a glass centrifuge tube (this volume is sufficient for late exponential/stationary phase cultures of Escherichia coli).
Note: Most plastic tubes are not compatible with chloroform, which will be added in a subsequent steps.
Notes:
If you wish to normalize your measured fatty acid concentrations to cell density, record the OD600 at this point.
If your culture is producing free fatty acids, a significant amount of foam may accumulate. It is necessary to collapse the foam, as a significant fraction of the free fatty acids present in the culture may be in the foam. This can be accomplished by the addition of antifoam followed by incubation in an 85 °C water bath for 5-10 min (Lennen et al., 2013). For example, 200 μl of a 1:10 dilution in ethanol of Antifoam 204 was added to collapse the foam of 50 ml shake flask cultures in Lennen et al. 2013.
To the cell culture, add ~50 μg of an appropriate internal standard. Over the course of this procedure, a fraction of this internal standard will be lost. From its known initial concentration and the final amount present, the measured concentration of each fatty acid methyl ester in the final extract can be used to determine the concentration of the corresponding acid in the culture. The choice of internal standard depends on which fatty acids are expected to be abundant in the samples to be analyzed. Often cells do not produce large amounts of odd-chain fatty acids. For example, E. coli grown on glucose does not produce large amounts of odd chain fatty acids, so heptadecanoic acid (C17:0) can be used as an internal standard.
If measuring membrane fatty acids or long chain free fatty acids (C16-C18 species), 5 μl of 10 mg/ml heptadecanoic acid dissolved in ethanol is a useful internal standard.
If the strain of interest is producing large quantities (0.5 g/L or higher) of medium chain free fatty acids (Lennen et al., 2013; Youngquist et al., 2012; Lenen et al., 2010), 50 μl of 10 mg/ml pentadecanoic acid (C15:0) dissolved in ethanol is a useful internal standard.
If only analyzing phospholipid species, a phospholipid internal standard, such as 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine in chloroform, can be used instead of a free fatty acid standard (Lennen and Pfleger, 2013).
Note that all values given for the volume/concentration of the internal standards may need to be adjusted for your particular system, and different standards may need to be used depending on the chain lengths of the fatty acids to be quantified. An internal standard should mimic the compounds to be quantified as closely as possible and should be added to the collected sample of culture in an amount which approximates (same order of magnitude) the concentration of the compounds it will be used to analyze.
5 μl of 10 mg/ml will correspond to a theoretical final concentration of 50 μg/ml methyl heptadecanoate if two 0.5 ml hexane extractions are performed in step 14. This is around the same order of magnitude as the C14:0, C16:0, and C16:1 fatty acids that are recovered from the membranes of wild-type E. coli.
Add 100 μl of glacial acetic acid to acidify the culture. Carefully vortex the sample to mix. This should be performed under a chemical fume hood.
Add 5 ml of 1:1 mixture by volume of chloroform and methanol with a glass pipette (chloroform will leach a variety of compounds out of a plastic pipette). Vortex thoroughly.
Notes:
This mixture may be stored at -80 °C and the extraction (detailed below) finished at a later date if desired.
If samples are frozen, each should be thawed to room temperature before moving on to step 5.
Centrifuge each sample for 10 minutes at 1,000 x g.
Using a vacuum line or aspirator, remove the upper aqueous layer and all cell debris at the interface.
Notes:
It is acceptable to aspirate a small amount of bottom chloroform layer if internal standards are present.
Prior to aspiration, ensure that proper receptacles are available for the waste generated by this process.
The chloroform layer can be stored at -80 °C at this point if necessary.
This step should be performed under a chemical fume hood!
Evaporate the chloroform extract under a nitrogen stream, leaving a dried residue in the tubes.
Notes:
There is usually some residual water that is difficult to remove after this step.
If tubes are being thawed out of the -80 °C freezer, you should re-aspirate any accumulated water droplets before proceeding to the evaporation step.
This step should be performed under a chemical fume hood!
Lyophilize the residue (30-60 min is sufficient) to remove any remaining water. All subsequent steps need to be performed with anhydrous samples.
For Total Fatty Acid Extraction (Free Fatty Acids and Bound Fatty Acid Species by Acid Catalysis):
To the dried extract, add 0.5 ml of anhydrous 1.25 M HCl in methanol. Cap tightly and heat at 50 °C overnight.
Notes:
Alternatively, the procedure can be performed at 75-80 °C for 1 h.
The elevated temperature builds up pressure in the sealed vial, so ensure you have tubes capable of withstanding these conditions if you do this (those described in the equipment section are adequate).
This step should be performed under a chemical fume hood!
Cool tubes to room temperature.
Add 0.5 ml of high resolution gas chromatography grade hexane. Take care to work rapidly to recap the tubes as hexane is highly volatile. This step should be performed under a chemical fume hood!
Add 5 ml of a 100 mg/ml NaHCO3 aqueous solution (this concentration is close to the solubility limit–it will dissolve after stirring overnight or warming the solution gently). Addition of bicarbonate quenches the acid-catalyzed reaction. This step should be performed under a chemical fume hood!
For Bound (Phospholipid) Fatty Acid Extraction by Base Catalysis:
To the dried chloroform extract, add 0.5 ml of 0.5 M sodium methoxide in methanol. This step should be performed under a chemical fume hood!
Incubate the reaction for 10 min at 50 °C, which is sufficient for trans-esterification of common phosphoplipids such as those found in E. coli.
Notes:
This reaction can also be used to trans-esterify other bound fatty acids, for example those found in triacylglycerols.
For guidelines on the incubation time for these reactions, which vary from the time required for phospholipids, consult a resource such as Lipid Analysis by William Christie (Christie and Han, 2010). Do not allow the reaction to proceed longer than necessary as too long a reaction time, and the presence of water, can result in esterification of free fatty acids.
The use of a free fatty acid internal standard as described previously can inform on whether only bound fatty acids are being methylated.
This step should be performed under a chemical fume hood!
Cool the tubes to room temperature. Quench the reaction by adding 0.1 ml of glacial acetic acid followed by 5 ml of deionized water. This step should be performed under a chemical fume hood!
Add 0.5 ml of high resolution gas chromatography grade hexane. This step should be performed under a chemical fume hood!
For Both Bound and Total Fatty Acid Extractions:
Vortex tubes thoroughly and centrifuge at room temperature for 10 minutes at 1,000 x g to create a stable interface. Note that the aqueous phase should be clear before proceeding.
Collect 0.4 ml of the hexane layer from the first extraction in an appropriate gas chromatograph vial. Be sure to cap the vial to prevent sample evaporation. This step should be performed under a chemical fume hood!
Add 0.5 ml of high resolution gas chromatography grade hexane to the methanol:water extract for a second extraction. Vortex thoroughly to mix and centrifuge at room temperature for 10 minutes at 1,000 x g to create a stable interface. This step should be performed under chemical fume hood!
Collect 0.5 ml of the top hexane layer and add to the hexane collected in step 14. The collected hexane layers are ready for GC analysis.
Notes:
Samples may need to be diluted in hexane if fatty acid concentrations are outside the range of standard calibration curves.
This step should be performed under chemical fume hood!
Analyze by gas chromatography with either a mass spectrometer or flame ionization detector. It is wise to randomize the run order of your samples to prevent the introduction of bias into your analysis. The following is an example of a typical gas chromatography method for bacterial lipids using an Agilent 7890 GC with a 30 m x 0.25 mm HP 5-ms capillary column (Lenen et al., 2010):
Inject 1 μl using a 1:10 split ratio of helium carrier gas (a lower split ratio, e.g. 1:100, can be used for more abundant species).
Oven temperature of 100 °C for 2 minutes.
Oven temperature of 150 °C for 4 minutes.
Ramp to 250 °C at a rate of 4 °C/min.
If you wish to know more about lipid analysis, an extremely useful reference is the website http://lipidlibrary.aocs.org/index.html. An example with real data is given in step 19.
To determine the concentrations of the fatty acid methyl esters of interest in your chromatography samples, you will need to run samples of these compounds at a variety of known concentrations (your external standards) spanning the full range of concentrations of each compound in the unknown samples. After running these samples, you will have a set of peak areas and corresponding concentrations for each compound. You may then construct a curve of best fit for each compound (including your internal standard), and calculate the concentration of each fatty acid methyl ester and the internal standard in your samples. These concentrations should be multiplied by the ratio of the theoretical to the actual internal standard concentrations to correct for sample lost during the extraction and methylation process. The concentrations in the bacterial culture may then be determined. Keep in mind that the concentration in units of mass for a fatty acid methyl ester and needs to be corrected to report the concentration of the corresponding fatty acid. It is wise to work in molar units during the analysis. As a rule of thumb, you should expect R2 values of about 0.98 to 0.99 or higher from your calibration curves.
Lipids were extracted from a bacterial culture expressing a plant thioesterase specific for 12 carbon (C12) saturated acyl-ACPs, resulting in the overproduction of C12 free fatty acids and, to a lesser extent, C14 free fatty acids. After adding 510 μg of pentadecanoic acid (C15) to 2.5 ml of a late stationary phase culture and performing the total fatty acid extraction protocol by acid catalysis as described above, the resulting FAMEs were analyzed by GC-MS. A total of 1 ml of hexane was used in the extraction. The amount of C12, C14, and C15 FAMEs present in the chromatography sample was determined by constructing calibration curves relating the peak area of each species to its concentration (column 2 of Table 1). If the conversion of C15 acid to its methyl ester and its subsequent extraction were perfect, there would have been 539.5 μg/ml of C15 methyl ester in the chromatography sample (calculated from the listed molecular weights). This fact was used to adjust the concentratiosn of C12 and C14 FAMEs (the measured concentrations were multiplied by the ratio 539.5/484.3). These corrected concentrations were then used to determine the fatty acid titer in the culture by converting to the acid concentration using the molecular weights and then dividing by the volume of the culture sample (2.5 ml).
Table 1. The titers of C12 and C14 fatty acids in a thioesterase expressing culture were measured by GC-MS using pentadecanoic acid (C15:0) as an internal standard. The concentration refers to the concentration in the sample analyzed by chromatography while titer refers to the concentration in the culture. Molecular weights were obtained from the NIST Chemistry Webbook.
Notes
Chloroform is toxic and should only be used under a fume hood. If chloroform comes into contact with latex or nitrile gloves, remove gloves immediately. Alternatively, use polyvinyl alcohol or SilverShield gloves when working with chloroform.
This procedure uses corrosive (glacial acetic acid and anhydrous hydrochloric acid in methanol), water-sensitive as well as flammable (methanol and hexane) chemicals. Sodium methoxide in methanol is flammable and highly toxic by inhalation, ingestion, and contact with skin. All should be handled according to common chemical safety practices.
Acknowledgments
The protocol described here is based on the work of Bligh and Dyer (1959). The protocol was developed with funding from the US Department of Energy, Great Lakes Bioenergy Research Center.
References
Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8): 911-917.
Christie, W. W. and Han, X. (2010). Lipid analysis: Isolation, separation, identification and lipidomic analysis, Oily Press Bridgewater, UK.
Lennen, R. M. and Pfleger, B. F. (2013). Modulating membrane composition alters free fatty acid tolerance in Escherichia coli. PLoS One 8(1): e54031.
Lennen, R. M., Politz, M. G., Kruziki, M. A. and Pfleger, B. F. (2013). Identification of transport proteins involved in free fatty acid efflux in Escherichia coli. J Bacteriol 195(1): 135-144.
Lenen, R. M., Braden, D. J., West, R. A., Dumesic, J. A. and Pfleger, B. F. (2010). A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol Bioeng 106(2): 193-202.
NIST Chemistry WebBook, NIST Standard Reference Database Number 69, Linstrom, P. J. and Mallard, W.G. (eds.) http://webbook.nist.gov/chemistry.
Youngquist, J. T., Lennen, R. M., Ranatunga, D. R., Bothfeld, W. H., Marner, W. D., 2nd and Pfleger, B. F. (2012). Kinetic modeling of free fatty acid production in Escherichia coli based on continuous cultivation of a plasmid free strain. Biotechnol Bioeng 109(6): 1518-1527.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Politz, M., Lennen, R. and Pfleger, B. (2013). Quantification of Bacterial Fatty Acids by Extraction and Methylation. Bio-protocol 3(21): e950. DOI: 10.21769/BioProtoc.950.
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Category
Biochemistry > Lipid > Lipid isolation
Microbiology > Microbial biochemistry > Lipid
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951 | https://bio-protocol.org/en/bpdetail?id=951&type=0 | # Bio-Protocol Content
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Peer-reviewed
Ovule Clearing Method for Solanaceous Species
AL Audrey Loubert-Hudon
DM Daniel P. Matton
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.951 Views: 10958
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Plant Journal Mar 2013
Abstract
Depending on the species, embryo sacs can be difficult to observe. Ovule clearing allows precise observation of whole tissues under Differential interference contrast (DIC) microscopy. The use of Methyl Salicylate as a clearing agent has proved to be particularly reliable for Solanaceous species.
Keywords: Solanum Ovule Reproduction Embryo sac Differential interference contrast (DIC)
Materials and Reagents
Flower buds
37% Formaldehyde
Glacial acetic acid
Methyl Salicylate
Ethanol (≥ 95%)
Fixative solution (FAA) (see Recipes)
Clearing solution (see Recipes)
Equipment
Forceps
Microscope with differential interference contrast (DIC)
Printed coated microscopy slides with wells (to avoid crushing the tissues)
Cover slips
Procedure
Carefully collect ovaries from flower buds or flowers at anthesis with precision forceps. During the process, keep ovaries and flowers on ice.
Figure 1. Organs and cells types of Solanaceous species’ flower and female gametophyte. a. Longitudinal sections of a Solanaceous flower. b. Transversal sections of a Solanaceous flower. c. Description of ovule cells types and their visualization in cleared ovules (false colors are used to emphasize cells types).
To facilitate penetration of the fixative and clearing solutions, the pericarp has to be removed. Under a magnifying glass, cut the top of the ovary’s pericarp with precision forceps or scalpel to allow its removal (see Figure 2).
Figure 2. Steps by steps procedure to remove the pericarp of Solanaceous species. a. Collect flowers at the desired stage of development and keep them on ice; b. Remove the upper part of the flower carefully by hand to keep only the ovary still attached to the pedicel; c. d. Cut the top of the ovary’s pericarp with precision forceps or scalpel to allow its removal; e. f. With the precision forceps, pinch under the bunch of ovules and put them directly in fixative solution.
A bunch of ovules can then be carefully taken and fixed in FAA solution overnight. Gentle agitation at 4 °C helps to homogenize the fixation, but is not crucial.
The day after, fixed ovaries are incubated in 95-100% ethanol for at least one hour, still with gentle agitation at room temperature.
The ovaries are then transferred in increasing ratio of Methyl Salicylate/EtOH solutions for 30 minutes each (1:3, 1:1, 3:1) at room temperature. Again, gentle agitation will help, but is not crucial.
The tissues are afterwards left in 100% Methyl Salicylate at room temperature. Make sure the tubes are well closed to avoid Methyl Salicylate evaporation.
The ovule can now be observed under differential interference contrast (DIC) microscopy. To avoid crushing the tissues, printed coated slides with wells are very useful. With precision forceps, carefully separate the ovules on the slide. Methyl Salicylate (100%) is used as mounting solution and slides are sealed with clear nail polish.
Recipes
Fixative solution (FAA)
1% formaldehyde
0.5% glacial acetic acid
50% ethanol
Note: It can be prepared in advance and stored at room temperature.
Clearing solution (see Procedure step 5, for concentration/ratio)
Methyl Salicylate
Ethanol
Acknowledgments
This protocol is adapted from Chevalier et al. (2013) and Estrada-Luna et al. (2004).
References
Chevalier, E., Loubert-Hudon, A. and Matton, D. P. (2013). ScRALF3, a secreted RALF-like peptide involved in cell-cell communication between the sporophyte and the female gametophyte in a solanaceous species. Plant J 73(6): 1019-1033.
Estrada-Luna, A., Garcia-Aguilar, M. and Vielle-Calzada, J. P. (2004). Female reproductive development and pollen tube growth in diploid genotypes of Solanum cardiophyllum Lindl. Sexual Plant Reproduction 17(3): 117-124.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Loubert-Hudon, A. and Matton, D. P. (2013). Ovule Clearing Method for Solanaceous Species. Bio-protocol 3(21): e951. DOI: 10.21769/BioProtoc.951.
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Category
Plant Science > Plant cell biology > Cell staining
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952 | https://bio-protocol.org/en/bpdetail?id=952&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
H2O2 Kill Assays of Biofilm Bacteria
Malika Khakimova
DN Dao Nguyen
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.952 Views: 10973
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Original Research Article:
The authors used this protocol in Journal of Bacteriology May 2013
Abstract
Ubiquitous in nature and often surface associated, biofilms cause numerous chronic human infections. Biofilms are structured multicellular bacterial communities where cells are entrapped in a polymer matrix. Bacteria growing as biofilms are characterized by marked tolerance to many biocides, including oxidants such as hydrogen peroxide. Hydrogen peroxide is both produced by host phagocytic cells, and used as an antimicrobial compound. Understanding biofilm tolerance to hydrogen peroxide is therefore relevant to the persistence of Pseudomonas aeruginosa in human infections (such as chronic Pseudomonas aeruginosa infections in cystic fibrosis airways) as well as in environmental settings (such as water pipes).
This protocol was developed to determine the tolerance of Pseudomonas aeruginosa biofilms to hydrogen peroxide (H2O2) killing. The bacteria are grown as colony biofilms on polycarbonate membranes, as previously described in Walters et al. 2003. The protocol may be adapted for other bacterial, with appropriate changes in H2O2 concentrations, since different bacterial species may be more or less susceptible to H2O2 than Pseudomonas aeruginosa.
Keywords: Pseudomonas aeruginosa Biofilms Oxidative stress Hydrogen peroxide Tolerance
Materials and Reagents
Phosphate buffered saline (PBS) solution (Sigma-Aldrich, catalog number: P4417-100TAB )
30% w/w Hydrogen peroxide solution (undiluted, as sold commercially) (RICCA Chemical, catalog number: 3821.7-32 )
Sodium thiosulfate solution (dissolved in ddH2O) (Sigma-Aldrich, catalog number: S8503 )
0.2 μM Polycarbonate 25 mm membranes (General Electric Company, catalog number: K02BP02500 )
P.aeruginosa strains in freezer stock
25% Lennox broth (LB) medium (Becton Dickinson and Company, DifcoTM, catalog number: 240230 ) (see Recipes)
25% LB agar plates (see Recipes)
Equipment
6-well and 96-well plates
Standard petri plates
Spectrophotometer (cuvette) (Thermo Fisher Scientific, model: GENESYS 10S UV-Vis )
Spectrophotometer (96-well plate) (Bio-Rad, model: 680 )
Cuvettes for OD600 reading
Shaking incubator at 37 °C, 250 rpm
Static incubator at 37 °C
Sterile glassware: 150 ml Erlenmeyer flasks, capped or foiled
1.5 ml and 2 ml microcentrifuge tubes
Source of UV irradiation
Sterile wire-loops (sterilized with 70% ethanol and flame)
Stainless steel forceps (sterilized with 70% ethanol and flame)
Procedure
Day 0. Streak P.aeruginosa cells from the freezer stock onto a LB agar place and incubate statically overnight at 37 °C.
Day 1. Pick 4-5 single colonies from the P.aeruginosa agar plate with a sterile wired-loop and inoculate 15 ml liquid LB medium in a 150 ml Erlenmeyer flask. Grow liquid bacterial cultures overnight for 16-18 hours at 37 °C, with shaking at 250 rpm.
Day 2. Gently place polycarbonate membranes on agar surface of fresh sterile 25% LB agar plates and sterilize the membranes by placing them under UV irradiation for 1 hour. Handle membranes carefully with sterile forceps and use the membranes immediately after sterilization. Use eyes and skin UV protective equipment. Use at least 3 membranes per strain per condition for adequate biological replicates, and up to 6 membranes may be placed on each agar plate.
Measure the OD600 of the overnight bacterial culture and dilute the bacterial suspension in LB medium to a starting concentration of 108 cells/ml. Depending on the bacterial strain used, the OD600 to CFU ratio will differ and needs to be determined for each strain: for example, for the PAO1 wild type strain, 108 cells/ml = ~OD600 0.1.
Spot 5 μl (5 x 105 cells) onto the sterile membranes and allow the liquid to be absorbed (10-20 minutes).
Incubate the colony biofilm on agar plates for 24 h at 37 °C.
Day 3. Using sterile forceps, gently lift the membranes off the agar surface and transfer them (cells side down) into 6-well plates filled with 2 ml of 25% LB liquid medium in each well. Make sure the membranes are spread flat (i.e. not rolled up) and biofilm cells are remain on the membrane.
For the H2O2 treated biofilms, add 30 μl H2O2 (150 mM) to each well in pulses every 10 minutes for 30 minutes (for a final concentration of 450 mM H2O2 per challenge). The pulsing is done to mimic a continuous exposure of cells to H2O2. In between H2O2 pulses, incubate cells at 37 °C without shaking. Include untreated controls that are challenged with PBS. Each condition should be done at least in triplicates.
After H2O2 or PBS challenge, add 0.2% sodium thiosulfate to all samples to neutralize any remaining H2O2. Add even when samples are only challenges with PBS as a control.
To determine the viable cell count in H2O2 or PBS treated biofilms, collect biofilm cells by transferring the membranes and 2 ml of liquid from each well into 2 ml microcentrifuge tubes. Membranes are moved by gently lifting and rolling them using sterile forceps, with biofilm cells facing inward. The entire membrane should be submerged in liquid. Ensure to sterilize forceps between membrane transfers. Vortex biofilms in microcentrifuge tubes at maximal speed for at least 1 minute to detach and resuspend cells. Additional pipetting up and down and vortexing may also necessary to make sure there are no cell clumps visible in the bacterial suspension.
Aliquot 100 μl of bacterial suspension into 96-well plate, serially dilute cells 1:10, then plate 100 μl of each dilution on LB agar plates for CFU count. Incubate CFU count plates at 37 °C overnight.
Day 4. Count CFU on LB agar plates and calculate the viable CFU per biofilm based on the dilution factors applied.
Determine hydrogen peroxide killing by comparing the viable CFU count in the PBS treated and the H2O2 treated conditions.
Recipes
25% LB medium
5 g LB powder medium per L
Dissolved in ddH2O and autoclaved
25% LB agar plates
25% LB medium with 1.5% agar
Dissolved in ddH2O and autoclave
Acknowledgments
We would like to acknowledge CIHR (MOP-102727 to DN) and the Burroughs Wellcome Fund (1006827.01 to DN) for funding. This protocol was adapted from the previously published paper Khakimova et al. (2013).
References
Khakimova, M., Ahlgren, H. G., Harrison, J. J., English, A. M. and Nguyen, D. (2013). The stringent response controls catalases in Pseudomonas aeruginosa and is required for hydrogen peroxide and antibiotic tolerance. J Bacteriol 195(9): 2011-2020.
Walters, M. C., 3rd, Roe, F., Bugnicourt, A., Franklin, M. J. and Stewart, P. S. (2003). Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 47(1): 317-323.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Khakimova, M. and Nguyen, D. (2013). H2O2 Kill Assays of Biofilm Bacteria. Bio-protocol 3(21): e952. DOI: 10.21769/BioProtoc.952.
Download Citation in RIS Format
Category
Microbiology > Microbial biofilm > Killing assay
Biochemistry > Other compound > Reactive oxygen species
Microbiology > Antimicrobial assay > Antibacterial assay
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953 | https://bio-protocol.org/en/bpdetail?id=953&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
H2O2 Kill Assays of Planktonic Stationary Phase Bacteria
Malika Khakimova
DN Dao Nguyen
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.953 Views: 9980
Download PDF
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Cited by
Original Research Article:
The authors used this protocol in Journal of Bacteriology May 2013
Abstract
Stationary phase bacteria are highly tolerant to hydrogen peroxide. This protocol was developed to test the susceptibility to hydrogen peroxide killing in different Pseudomonas aeruginosa strains. This assay provides a reliable way to measure killing of stationary phase bacterial cells to hydrogen peroxide and can be adapted to test other oxidants.
Materials and Reagents
Phosphate buffered saline (PBS) solution (Sigma-Aldrich, catalog number: P4417-100TAB )
30% w/w Hydrogen peroxide stock solution (RICCA Chemical, catalog number: 3821.7-32 )
Sodium thiosulfate solution (dissolved in ddH2O) (Sigma-Aldrich, catalog number: S8503 )
P.aeruginosa strains in freezer stock
25% Lennox broth (LB) medium (Becton Dickinson and Company, DifcoTM, catalog number: 240230 ) (see Recipes)
LB agar plates (see Recipes)
Equipment
96-well plates
Standard petri plates
Spectrophotometer (cuvette) (Thermo Fisher Scientific, model: GENESYS 10S UV-Vis )
Spectrophotometer (96-well plate) (Bio-Rad Laboratories, model: 680 )
Cuvettes for OD600 reading
Shaker-incubator at 37 °C, 250 rpm
Static incubator at 37 °C
Sterile glassware: 150 ml Erlenmeyer flask, capped or foiled
Sterile 15 mm glass test tubes and plastic caps
Sterile wire-loops (sterilized with 70% ethanol and flame)
Procedure
Day 0. Streak P.aeruginosa cells from the freezer stock onto a LB agar place and incubate statically overnight at 37 °C.
Day 1. Pick 4-5 single colonies from the P.aeruginosa agar plate with a sterile wired-loop and inoculate 15 ml liquid LB medium in a 150 ml Erlenmeyer flask. Grow liquid bacterial cultures overnight for 16-18 hours at 37 °C, with shaking at 250 rpm.
Day 2. Inoculate 15 ml liquid LB medium in a 150 ml flask with 1:100 of overnight bacterial culture. Grow cells to for 16-18 hours at 37 °C, with shaking at 250 rpm.
Day 3. Determine the OD600 of the culture and dilute the bacterial suspension to a starting concentration of ~2.5 x 106 cells/ml (in total volume 1 ml LB). Depending on the bacterial strain used, the OD600 to CFU ratio will differ and needs to be determined for each strain: for example, for the PAO1 wild type strain, 108 cells/ml = ~OD600 0.1.
To confirm the correct starting bacterial density (at ~2.5 x 106 cells/ml), aliquot 100 μl of the above bacterial suspension into 96-well plate. Serially dilute cells 1:10 in PBS to approximately ~2.5 x 102 cells/ml, then plate 100 μl on LB agar plates for CFU count. This will also be the CFU count for time zero measurement.
Set up ~2.5 x 106 cells/ml x 1 ml per sample in sterile glass tubes, with at least 3 replicates per strain per condition. For H2O2 treated samples, add H2O2 (1 mM (2 μl) to 5 mM (10 μl) or other desired final concentration) to each samples in test tubes. Include untreated controls that are challenged with PBS. Each condition should be done at least in triplicates. Incubate cells for 2 hours with shaking at 250 rpm at 37 °C.
After H2O2 or PBS challenge, add 0.2% sodium thiosulfate to all samples to neutralize any remaining H2O2. Add even when samples are only challenges with PBS as a control.
To determine the viable cell count in H2O2 or PBS treated samples, aliquot 100 μl of bacterial samples into 96-well plate, serially dilute cells 1:10, then plate 100 μl of each dilution on LB agar plates for CFU count. Incubate CFU count plates at 37 °C overnight.
Day 4. Count CFU on LB agar plates and calculate the viable CFU per biofilm based on the dilution factors applied.
Determine hydrogen peroxide killing by comparing the viable CFU count in the PBS treated and the H2O2 treated conditions.
Recipes
25% LB medium
5 g LB powder medium per L
Dissolved in ddH2O and autoclaved
LB agar plates
LB medium with 1.5% agar
Dissolved in ddH2O and autoclaved
Acknowledgments
We would like to acknowledge CIHR (MOP-102727 to DN) and the Burroughs Wellcome Fund (1006827.01 to DN) for funding. This protocol was adapted from the previously published paper Khakimova et al. (2013).
References
Khakimova, M., Ahlgren, H. G., Harrison, J. J., English, A. M. and Nguyen, D. (2013). The stringent response controls catalases in Pseudomonas aeruginosa and is required for hydrogen peroxide and antibiotic tolerance. J Bacteriol 195(9): 2011-2020.
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Category
Microbiology > Antimicrobial assay > Killing assay
Biochemistry > Other compound > Reactive oxygen species
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954 | https://bio-protocol.org/en/bpdetail?id=954&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Bacterial Counts in Spleen
Elías Barquero-Calvo
CC Carlos Chacón-Díaz
EC Esteban Chaves-Olarte
EM Edgardo Moreno
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.954 Views: 25496
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Original Research Article:
The authors used this protocol in PLOS Pathogens Feb 2013
Abstract
Bacterial loads can be determined as colony forming units (CFU) at any point of the infection by culturing spleen homogenates on agar plates. This is a reliable method for comparing the kinetics of infection in various mouse strains, estimating the virulence of different bacterial mutants or isolates and for vaccine testing and vacine estandarization. Although this method has been designed to recover Brucella or Salmonella organisms from spleen, the procedure may be applicable for other bacteria such as Listeria and Mycobacterium as well as to count bacterial loads in other organs such as liver or lymph nodes.
Keywords: Spleen CFU Bacteria Mouse
Materials and Reagents
Sterile plastic bags, 30 g capacity (~30 ml) (Whirl-Pak® Write-On Bags) (Nasco, catalog number: B01067WA )
70% Ethanol (Merck KgaA, Emsure®, catalog number: 1009834000 )
Latex examination gloves (Dermagrip®, catalog number: D1402-14 )
Pyrogen-free Type 1+ grade distilled water (Milli-Q Direct 8 and 16 Ultrapure Water System)
Dulbecco′s Phosphate Buffered Saline (Gibco, catalog number: 21300-058 )
Tween® 20 (Sigma-Aldrich, catalog number: P5927 )
Tryptone Soya Agar (Oxoid Limited, catalog number: CM0131 ) (see Recipes), or other media according to culture bacterial requirements
PBS 0.1% Tween 20 (see Recipes)
Equipment
24-well plates, low binding cell (Sigma-Aldrich, catalog number: Z721077 )
Beaker (100 ml)
Glass alcohol burner (WHEATON, catalog number: WHE-237070 )
Surgical instruments for mice (Mouse Surgical Kit) (Kent Scientific, model: INSMOUSEKIT )
Drigalski spatula (Thermo Fisher Scientific, catalog number: NC0242405 )
Micropipettes (10-100 μl and 100-1,000 μl)
Micropipette tips (100 μl and 1 ml)
1.5 ml eppendorf tubes (Eppendorf, catalog number: 0 22363204 )
Tuberculin plastic syringe (1 ml) with 25-27 g needle gauge
Plastic petri dishes (90 x 15 mm)
Disposable medical plastic gloves (Lab Depot, catalog number: 266-P )
Small sharp scissors and forceps
Water purification system (Milli-Q Direct 8 and 16 Ultrapure Water System) (EMD Millipore, model: ZR0Q00800 )
Bacterial incubator (Forma® Direct Heat CO2 Incubator) (Thermo Fisher Scientific, model: 310 )
Autoclave (Yamato Scientific, model: SQ-500C )
Analytical balance electronic digital (± 0.0001 g)
Laminar flow cabinet (Esco Global, Labculture® Class II, Type A2, model: LA2-3A2 )
Procedure
Spleen extraction
Latex examination gloves should be used throughout the entire procedure.
Mice (Note 1) are infected by the corresponding via (e.g. intraperitoneally, intravenously, subcutaneously) using the recommended bacterial dose (e.g. 102, 103, 104) depending on the assay and virulence of the bacteria employed. Intraperitoneal infection is carried out with tuberculin 1 cc syringe, 25-27 g needle and maximum volume of 20 ml/kg.
http://www.procedureswithcare.org.uk/intraperitoneal-injection-in-the-mouse
Weight sterile plastic Whirl-Pak Bags (one per each mouse spleen) to the mg level.
Spleen must be obtained immediately after sacrifice (Note 2). Following the corresponding infection period (e.g. 3-15 days), mice are killed by cervical elongation keeping the appropriate ethical protocols and regulations (e.g. cervical dislocation, CO2) (Nagy et al., 2006) (Note 3).
After killing, spray the mouse exhaustively with 70% alcohol, and locate on a clean or sterile surface, within a safety vertical laminar flow hood.
With small sharp scissors make a small cut into the skin below the belly bottom. Then open the entire abdominal cavity and cut the peritoneum and expose the spleen (situated in the left superior abdominal quadrant of the mouse) or the required organ, following recommended necropsy protocols (Covelli, 2013 http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php) (Note 4) (Figure 1).
Figure 1. Spleen extraction indicating: A. position of the mouse for necropsy, B. opening of the mouse skin, C. opening of the abdominal wall and D. spleen extraction (pointed with white arrow). Photographs taken from Covelli, 2013. For details, this work may be consulted at: http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php (date consulted: 09/30/2013).
Use small forceps to hold the spleen and then cut the hilum together with the gastrosplenic ligament to remove the spleen and locate inside a pre-weighted sterile plastic Whirl-Pak Bag (Note 5) (Figure 1).
Weight the spleens inside the bags using a 4 digit scale balance. To obtain the spleen weight, subtract the value of the empty preweighted plastic bag.
Spleen homogenization
Add 9 parts of PBS containing 0.1% Tween 20 per g of spleen (dilution 1:10), assuming that the volume of 1 g of spleen corresponds to 1 ml of PBS (e.g. 0.5 grams of spleen and 4.5 ml of PBS 0.1% Tween 20).
Note: Homogenization is easier if a small volume is used at the beginning of the homogenization procedure and then, the remaining volume is completed to reach 1:10 dilution.
Spleen homogenization is proficiently carried out by squeezing the organ inside bag by hand.
Note: In order to release the intracellular bacteria, spleen cells are disrupted by squeezing the spleen tissue in PBS containing 0.1% Tween 20.
After homogenization of the samples, and depending on expected bacterial spleen colonization, decimal (or the required) dilutions are performed with 1x PBS (on 1.5 ml eppendorf tubes or 24 well plates).
Note: Open the plastic bag carefully and widely (using steel wires) to avoid contamination of the pipette. For infections using 1 x 106 CFU of bacteria and ranging from 1-30 days, plate 102-105 dilutions.
Plating
Two plating methods are suggested:
Method A
Dispense three separate 20 μl drops of each dilution on Tryptone Soya Agar (or the recommended media for growing the tested bacterium) plates using two plates per sample (Figure 2A). Then, let the drops to be absorbed in the agar surface (do not spread out the drops) prior incubation (5-10 minutes).
Method B
Dispense one 100 μl in each agar plate and distribute the sample with a sterile Drigalski spatula until the inoculum is fully dispersed on the agar surface using two plates per sample (Figure 2B).
Notes:
Method A is quicker and fewer agar plates are required since four dilutions can be positioned on each plate. In addition, up to three drops can be put for each dilution, lessening counting errors. A minimum of 500 bacteria per g of spleen can be detected if 101dilution is plated.
Method B is slower and increases plate consuming but is more sensitive when low bacterial loads are expected. Colony counting is easier since an entire plate is used per dilution. A minimum of 100 bacteria per g of spleen can be detected if 101dilution is plated.
Plates are incubated under the required conditions for the bacterial strain (e.g. 37 °C under 5% CO2 atmosphere for 2-3 days) until colony forming units (CFUs) are evident.
Count CFU in the various dilutions. Only use the plates (and the corresponding dilutions) in which separate colonies are obtained (10-30 CFUs in Figure 2A or 30-300 CFUs in Figure 2B).
Calculate spleen bacterial loads by multiplying CFUs to the corresponding dilution and by 50 in method A or by 10 in method B, respectively.
CFUs means can be calculated either by CFUs/spleen (total CFUs recovered) or alternatively by CFUs/gram of spleen (total CFUs recovered divided by total grams of spleen). While the first method does not take into consideration spleen inflammation (size), the second method corrects for spleen swelling. In either case, it is always recommended to plot the spleen weight in a separate graph.
Figure 2. Sample plating methods
Notes
Preferably, female mice (male mice from different litters have the tendency to fight) with weights ranging between 18-22 grams. Weight/age ratio varies depending upon the strain of mice. Older mice have the tendency of display more weight variation. Differences in weights or ages should be avoided. It is recommended to work with groups of four mice in tandem. Mice should be in good health conditions before the experiment. In the case of using immunosuppressed mice, it is recommended to include a group of non-infected negative control mice to check for previously acquired bacterial or yeast infections.
Bacterial counts in organs after natural death or after a protracted period of time are not recommended, due to microorganism contamination during the process of tissue decay.
Other methods used for sacrifice are CO2 affixation or overdose of anesthesia with components such as isoflurane (inhaled > 15%) or propofol (intravenously > 50 mg/kg).
Avoid the skin hair of the mouse to become in contact with the peritoneal cavity.
The spleen is easily detached without abundant hemorrhage. Surgical instruments must be sterilized with alcohol and flamed prior spleen manipulation to avoid contamination. External fat should be removed from the spleen to avoid extra weight errors.
Recipes
Tryptone Soya Agar
Suspend 40 grams of medium in 1 L Pyrogen-free Type 1+ grade distilled water
Heat with frequent agitation and boil for 1 minute to completely dissolve the medium
Autoclave at 121 °C for 15 min
PBS 0.1% Tween 20
Dissolve the following in 800 ml Pyrogen-free Type 1+ grade distilled water H2O
8 g of NaCl
0.2 g of KCl
1.44 g of Na2HPO4
0.24 g of KH2PO4
1 ml Tween 20
Adjust pH to 7.4
Adjust volume to 1 L with additional Pyrogen-free Type 1+ grade distilled water H2O
Sterilize by autoclaving
Acknowledgments
We thank María-Jesús Grilló for helping in the standardization of this technique. This protocol was adapted from the following original published papers Barquero-Calvo et al. (2007) and Nagy et al. (2007), and from Covelli (2013). This work was funded by grants from FIDA-2006, FS-CONARE (UNA/UCR), MICIT/CONICIT (FI-487-09), 8-N-2005 and B/3456-1 (NeTropica), B/3456-2 (IFS), AGL2004-01162/GAN (Spain), CNRS and INSERM (France) and MASTERSWITCH projects (European Communities).
References
Barquero-Calvo, E., Chaves-Olarte, E., Weiss, D. S., Guzman-Verri, C., Chacon-Diaz, C., Rucavado, A., Moriyon, I. and Moreno, E. (2007). Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection. PLoS One 2(7): e631.
Covelli, V. (2013). Guide to the Necropsy of the Mouse.
http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php
Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R. (2006). Quick and humane sacrifice of a mouse by cervical dislocation. CSH Protoc 2006(1).
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Barquero-Calvo, E., Chacón-Díaz, C., Chaves-Olarte, E. and Moreno, E. (2013). Bacterial Counts in Spleen. Bio-protocol 3(21): e954. DOI: 10.21769/BioProtoc.954.
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Category
Immunology > Animal model > Mouse
Cell Biology > Tissue analysis > Tissue isolation
Microbiology > Microbe-host interactions > Ex vivo model
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955 | https://bio-protocol.org/en/bpdetail?id=955&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Dissection of Different Areas from Mouse Hippocampus
FS Faraz A. Sultan
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.955 Views: 27046
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Nov 2012
Abstract
The hippocampus modulates a number of modules including memory consolidation, spatial navigation, temporal processing and emotion. A banana-shaped structure, the hippocampus is constituted of morphologically distinct subregions including the dentate gyrus, CA3 and CA1 (here, we do not distinguish the “hippocampus proper” which consists only of CA1, CA3 and smaller CA2 and CA4 areas, from the “hippocampal formation,” composed of these in addition to the dentate gyrus and subiculum). Distinct cell types give rise to unique axonal fiber pathways in the dentate gyrus, CA3 and CA1 subregions; accordingly, these areas may exhibit differential molecular profiles in response to a number of behavioral paradigms and pharmacological and genetic treatments. It is therefore in the interest of the investigator to dissect a specific subregion from the whole hippocampus. Here we outline a protocol for subregion-specific dissection from the adult mouse.
Keywords: Hippocampus Memory Learning CA1
Materials and Reagents
Microspatula (Thermo Fisher Scientific, catalog number: 21-401-10 )
Short microspatula (Thermo Fisher Scientific, catalog number: 21-401-15 )
Filter paper (11 cm) (Thermo Fisher Scientific, catalog number: 09-795D )
WypAll or KimWipes
Ice buckets
Cloth diapers
Dry Ice
Biohazard waste bags
Adult laboratory mice
10x Stock Cutting Solution (see Recipes)
1 M KCl (see Recipes)
1 M MgCl2 (see Recipes)
1 M CaCl2 (see Recipes)
1x Cutting Solution (make from 10x stock) (see Recipes)
Equipment
Surgical Scissors (Fine Science Tools, catalog number: 14130-17 )
Fine Scissors (Fine Science Tools, catalog number: 14094-11 )
Forceps (Fine Science Tools, catalog number: 11506-12 )
Scalpel handle (Fine Science Tools, catalog number: 10003-12 )
Scalpel blades #15 (Fine Science Tools, catalog number: 10015-00 )
Single edge razor blades
Petri dishes (100 mm x 15 mm) (Sigma-Aldrich, catalog number: CLS3160101 )
Transfer pipettes (Thermo Fisher Scientific, catalog number: 13-711-7M )
20 ml beaker (Thermo Fisher Scientific, catalog number: 02-539-1 )
600 ml beaker (Sigma-Aldrich, catalog number: CLS1000600 )
1 L bottle (Sigma-Aldrich, catalog number: CLS13951L )
Nuclease-free microtubes (1.5-1.7 ml)
Plastic spoon
Microscope
Pressurized oxygen tank (95% O2/5% CO2) with tubing
Procedure
Place container of 1x Cutting Solution in a filled ice bucket, and transfer ~15 ml of the solution into a 20 ml beaker also on wet ice.
Oxygenate the solutions in both the bottle and beaker for at least 20 min before beginning (see below).
Surround bench with cloth diapers, and secure a biohazard waste bag near the work bench.
Place all dissection tools except razor blades and scalpel into a 600 ml beaker containing sterile water and WypAll or KimWipe cloths submerged in the water. Place the tools facing down on the cloths so as to minimize direct contact with the glass beaker.
Place labeled microtubes in a bucket containing dry ice (blue bucket above). Likewise, place one piece of a petri dish on the dry ice and let cool.
Place small ice bucket containing ice under microscope (purple bucket above).
Fill one ice bucket with ice and secure the other piece of the petri dish on the ice so that the edges are facing down. Place one piece of filter paper on the dish.
Immediately before beginning, transfer enough cutting solution from the 20 ml beaker to the filter paper and spread evenly.
Place a new cloth diaper over the bench before each dissection. Place the mouse on the diaper, maintaining a grip over the tail with the right hand (for right-handed individuals).
Rapidly secure the mouse with the left hand such that the scruff posterior to the rodent’s head is firmly held between the left thumb and proximal forefinger. The mouse should be firmly in the experimenter’s control at this point. Transfer the base of the tail from the right hand to the left hand so that it is secured between the distal ring and little fingers.
Place smooth end of large scissors just behind the skull and hold down firmly with the right hand. Rapidly pull on the tail with the left hand until the rodent skull is dislocated from the spinal cord.
After cervical dislocation, decapitate with the same scissors, being sure to cut just behind the skull. If the cut is too far posterior, excess tissue will occlude the foramen magnum.
While gently pulling the scalp to lateral sides, cut scalp skin from between the rodent’s eyes down the midline using a razor blade.
Place one tip of the fine scissors into the foramen magnum and cut laterally into the skull. Repeat for the other side. Gently cut from the same cavity up the midline towards the nose, trying to keep the end of the scissors as superficial as possible so as not to perturb the brain.
Make small cuts from the midline incision near the eyes laterally.
Use forceps to apply gentle tangential, lateral pressure to either of the newly formed skull flaps. Repeat for the remaining side. If force is properly applied, the skull should be fully removed and the brain exposed. If a piece of skull breaks off before the respective hemisphere is exposed, discard it and use forceps again to coerce the remaining flap to the side. In this case, be sure to minimize contact between the forceps tips and brain tissue.
Use longer microspatula to gently transfer the brain to the 20 ml beaker of oxygenated cutting solution. This involves tearing cranial nerve fibers near the base of the brain.
Use the spoon to transfer the brain to the wetted filter paper, and hemisect the brain using a clean razor blade. Transfer either hemisphere back to the 20 ml beaker.
Using the two short microspatulas, remove the hippocampus: Anchor one spatula tip just over the cerebellum near the junction with the cortex. Place the other spatula tip near the same junction and peel the cortical hemisphere laterally in a gentle manner. Periodically use the transfer pipette to apply fresh cutting solution to the tissue.
Once the cortical hemisphere is fully peeled laterally, the hippocampus should be exposed. While anchoring the brain with one spatula tip, place the other just under the caudal tip of the hippocampus. Carefully apply pressure to the medial white matter tracts with the anchoring spatula while moving the second spatula tip slightly anteriorly and laterally. “Scoop” or “roll” the hippocampus laterally with that spatula tip. If done properly, the hippocampus should land on the filter paper with “glossy” end up. Repeat for the second hemisphere.
Once both hippocampi are removed and placed on the filter paper, transfer it along with the petri dish piece to the ice bucket underneath the microscope. Align each hippocampus so that the anterior end (slightly thinner than the posterior end – this is more easily seen by observing the hippocampus from the side rather than the top) is facing upward.
Use the scalpel to make small vertical incisions in both the anterior and posterior tips of the hippocampus. This will create flat, blunt tips.
Make a similar incision about 1/3rd or 1/4th of the way from the anterior to posterior end while observing through the microscope. Carefully “stand” the small segment of the tissue above the blade on the face created earlier (right figure below – CA1 is on top). Be sure to apply fresh cutting solution but only in small quantities – excess solution will blur the distinctions of the hippocampal subregions.
Using the scalpel, dissect out area CA1, CA3 or dentate gyrus (DG) with one curved cut downwards through the tissue (see diagram below for approximate dissection guidelines for a right hippocampus cross-section). Immediately transfer the dissected tissue with the scalpel to the petri dish piece on dry ice. The tissue should freeze very rapidly.
Repeat step 23 with the remaining piece of tissue, keeping in mind that CA1 ends approximately 2/3rds of the way from the anterior to posterior end. Depending on the thickness of pieces cut, a total of 2-4 segments will have been created. Work quickly and minimize overall disruption of brain tissue.
Once all pieces of tissue are frozen on dry ice, use the scalpel blade to detach the tissue from the petri dish and place in a labeled microtube. It is often convenient to “stack” tissue on the petri dish as it is collected. This way, one larger piece will be created, and it can be dislodged more easily than multiple small pieces of frozen tissue.
Store microtubes at -80 °C until tissue is ready for processing.
Recipes
10x Stock Cutting Solution
For 1 L stock, mix the following in a graduated cylinder and bring up to 1 L with sterile Milli-Q water:
73.00 g NaCl
1.72 g Sodium phosphate (monobasic, monohydrate)
21.00 g Sodium bicarbonate
18.10 g D-Glucose
Sterile filter with vacuum filtration, and stored at 4 °C
1 M KCl
Dissolve 14.91 g KCl in Milli-Q water and bring up to a volume of 200 ml
Sterile filter and stored at 4 °C
1 M MgCl2
Dissolve 40.66 g MgCl2 hexahydrate in Milli-Q water and bring up to a volume of 200 ml
Sterile filter and stored at 4 °C
1 M CaCl2
Dissolve 29.40 g CaCl2 dihydrate in Milli-Q water and bring up to a volume of 200 ml
Sterile filter and stored at 4 °C
1x Cutting Solution
For 1 L solution:
Mix 100 ml 10x stock solution
120 mg sodium L-ascorbate
3 ml 1 M KCl stock, bring up to 900 ml with Milli-Q water
Begin oxygenation with 95% O2/5% CO2 for at least 20 minutes
Add 7 ml MgCl2 and 0.5 ml CaCl2 and continue oxygenation
Confirm that pH is in physiological range ~7.2-7.4
Bring up to a final volume of 1 L with Milli-Q water
(Optional) Confirm that osmolarity is ~315-325 mOsm
Sterile filter, keep container on ice and bring to experiment setup
Acknowledgments
This protocol was adapted from a previously published paper: Levenson et al. (2006). This work was supported by the NIH (MH095270, MH57014, AG031722, NS057098, P30 NS47466), the Ellison Medical Foundation, and the McKnight Brain Research Foundation.
References
Levenson, J. M., Roth, T. L., Lubin, F. D., Miller, C. A., Huang, I. C., Desai, P., Malone, L. M. and Sweatt, J. D. (2006). Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J Biol Chem 281(23): 15763-15773.
Miller, C. A. and Sweatt, J. D. (2007). Covalent modification of DNA regulates memory formation. Neuron 53(6): 857-869.
Sultan, F. A., Wang, J., Tront, J., Liebermann, D. A. and Sweatt, J. D. (2012). Genetic deletion of Gadd45b, a regulator of active DNA demethylation, enhances long-term memory and synaptic plasticity. J Neurosci 32(48): 17059-17066.
Sweatt, J. D. (2009). Mechanisms of Memory. Academic Press.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Sultan, F. A. (2013). Dissection of Different Areas from Mouse Hippocampus. Bio-protocol 3(21): e955. DOI: 10.21769/BioProtoc.955.
Sultan, F. A., Wang, J., Tront, J., Liebermann, D. A. and Sweatt, J. D. (2012). Genetic deletion of Gadd45b, a regulator of active DNA demethylation, enhances long-term memory and synaptic plasticity. J Neurosci 32(48): 17059-17066.
Download Citation in RIS Format
Category
Neuroscience > Behavioral neuroscience > Cognition
Cell Biology > Tissue analysis > Tissue isolation
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956 | https://bio-protocol.org/en/bpdetail?id=956&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Adhesion of Moraxella catarrhalis to Respiratory Tract Epithelial Cells
Stefan P.W. de Vries
Hester J. Bootsma
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.956 Views: 9063
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Microbiology Jan 2013
Abstract
Moraxella catarrhalis is a human-restricted pathogen that is responsible for respiratory tract infections such as childhood otitis media (OM) and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Successful colonization and infection by M. catarrhalis depends on its ability to attach to the respiratory tract mucosal epithelium. This protocol describes a method to measure adherence of M. catarrhalis to epithelial cell lines in vitro.
Keywords: Moraxella catarrhalis Epithelial cells Adhesion Respiratory tract Pathogenesis
Materials and Reagents
Human pharyngeal epithelial cell line Detroit 562 (ATCC, catalog number: CCL-138 )
Type II alveolar epithelial cell line A549 (ATCC, catalog number: CCL-185 )
DMEM + GlutaMAXTM-I (Life Technologies, Invitrogen™, catalog number: 31966-047 )
Fetal calf serum (FCS) (Greiner Bio-One GmbH, catalog number: 758093S5403 )
Trypsin-EDTA (0.25%-1 mM) (Life Technologies, catalog number: 25300-054 )
Brain heart infusion (BHI) (Becton Dickinson and Company, catalog number: 237500 ) broth and BHI agar plates
Antibiotics: spectinomycin or kanamycin (Merck KgaA, Calbiochem, catalog numbers: 567570-10 and 420311-5 )
Bovine skin gelatin (Sigma-Aldrich, catalog number: G9382-100G )
Glycerol (Merck KgaA, catalog number: 1.04092.1000)
PBS without Ca2+ and Mg2+ (Westburg BV, catalog number: BE17-516F/12 )
Saponin (Sigma-Aldrich, catalog number: 47036-50G-F )
Equipment
24-well tissue culture plate (Falcon®, catalog number: 353407 )
75 cm2 culture flask
Centrifuge
CO2 incubator
Benchtop Incubator Shaker
Procedure
The human pharyngeal epithelial cell line Detroit 562 and the type II alveolar epithelial cell line A549 were routinely grown in DMEM with GlutaMAXTM-I and 10% FCS at 37 °C and 5% CO2.
For passage or seeding purposes epithelial cells were washed once in 10 ml PBS and detached using trypsin-EDTA as follows: add 3 ml Trypsin-EDTA per 75 cm2 culture flask, incubate 5 min at 37 °C and 5% CO2 until cells have detached from the bottom, collect cells with 7 ml culture medium, count cells, spin required amount for 5 min at 400 x g, resuspend pellet in required volume of culture medium.
Two days prior to the adherence assay, 2 x 105 Detroit 562 cells per well were seeded into a 24-well tissue culture plate, and after one day, the growth medium was refreshed.
A549 cells were seeded into 24-well tissue culture plates one day prior to the assay at 4 x 105 cells.
For both cell lines, monolayers of approximately 1 x 106 cells per well were used for adherence assays.
M. catarrhalis strains were inoculated on brain heart infusion (BHI) plates (supplemented with antibiotics when required) and grown overnight at 37 °C in an atmosphere containing 5% CO2.
Bacteria were harvested from plate and resuspended in PBS supplemented with 0.15% gelatin (PBS-G).
This suspension was used to inoculate BHI broth to an OD620 nm of ~ 0.05 and grown at 37 °C at 200-250 rpm until OD620 nm of 1.0 to 1.2. Subsequently, glycerol was added to a final concentration of 20%, and 1-ml aliquots were stored at -80 °C.
Before each assay, bacteria were thawed on ice, washed once in 1 ml DMEM with GlutaMAXTM-I and 1% FCS (infection medium) and resuspended in the infection medium to 1 x 107 CFU ml-1.
Epithelial cells were washed twice with 1 ml PBS, infected with 1 ml of the M. catarrhalis suspension (multiplicity of infection, 10 bacteria per cell), centrifuged for 5 minutes at 200 x g to facilitate contact between bacteria and cells, and incubated 1 h at 37 °C in a 5% CO2 environment.
Non-adherent bacteria were removed by 3 washes with 1-ml PBS (PBS was added and subsequently carefully aspirated, no agitation). Detroit 562 or A549 cells were detached and lysed by addition of 1 ml 1% saponin in PBS-G followed by incubation at 37 °C and 5% CO2 for 10 min.
CFUs were enumerated by plating 10-fold serial dilutions on BHI plates supplemented with the appropriate antibiotics. The percentage adherence of the mutants was calculated as the fraction of the inoculum that bound to the Detroit 562 cells.
Acknowledgments
This protocol was published in: de Vries et al. (2013). This study was financially supported by Vienna Spot of Excellence (VSOE) grant (ID337956).
References
de Vries, S. P., Burghout, P., Langereis, J. D., Zomer, A., Hermans, P. W. and Bootsma, H. J. (2013). Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions. Mol Microbiol 87(1): 14-29.
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© 2013 The Authors; exclusive licensee Bio-protocol LLC.
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Category
Microbiology > Microbe-host interactions > In vitro model
Cell Biology > Cell structure > Cell adhesion
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957 | https://bio-protocol.org/en/bpdetail?id=957&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles
Stefan P.W. de Vries
Hester J. Bootsma
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.957 Views: 8689
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Microbiology Jan 2013
Abstract
Phenotypes observed for certain Moraxella catarrhalis wild-type strains or mutants may be caused by a variety of factors including alteration of outer membrane protein composition. Examination of the outer membrane protein profiles may be a valuable tool to identify changes in outer membrane compositions of these strains. Here we describe a method to isolate and analyse M. catarrhalis fractions highly enriched for membrane proteins.
Keywords: Moraxella catarrhalis Outer membrane vesicles Pathogenesis Virulence
Materials and Reagents
Brain heart infusion (BHI) (Becton Dickinson and Company, catalog number: 237500 ) broth and BHI agar plates
Antibiotics
e.g. spectinomycin or kanamycin (Merck KgaA, Calbiochem, catalog numbers: 567570-10 and 420311-5 )
PBS
Bovine skin gelatin (Sigma-Aldrich, catalog number: G9382-100G )
ReadyPrep Protein Extraction Kit (membrane I) (Bio-Rad, catalog number: 1632088 )
Glass beads, acid-washed (150-212 μm) (Sigma-Aldrich, catalog number: G1145 )
2D-Quant Kit (General Electric Company, catalog number: 80-6483-56 )
Mini-protean TGX precast gels, 4-15% (Bio-Rad, catalog number: 456-1083 )
Colloidal Coomassie staining (Pink et al., 2012)
Equipment
CO2 incubator
Benchtop Incubator Shaker
Centrifuge
TissueLyser LT (QIAGEN, model: 85600 )
Procedure
M. catarrhalis strains were inoculated on brain heart infusion (BHI) plates (supplemented with antibiotics when appropriate), and grown overnight at 37 °C in an atmosphere containing 5% CO2.
Bacteria were harvested from plate and resuspended in PBS supplemented with 0.15% gelatin (PBS-G). This suspension was used to inoculate BHI broth to an OD620 nm of ~ 0.05 and grown at 37 °C at 200-250 rpm until OD620 nm of 1.0 to 1.2 (mid-log). Use of different growth media is possible, but may affect outer membrane protein profiles. Always use the same growth media when comparing outer membrane protein profiles of different strains.
Subsequently, bacteria were harvested by centrifugation for 10 minutes at 3,200 x g.
Outer membranes were isolated using the ReadyPrep Membrane I kit according to manufacturer’s instructions.
During the lysis procedure, 50 mg acid-washed glass beads (150-212 μm) were added for homogenization with the TissueLyser LT. The TissueLyser was operated 5 times for 1 minute at 50 Hz.
Suspensions were chilled on ice for 1 minute between every TissueLyser step.
Protein quantification was performed using the 2D-Quant Kit according to manufacturer’s instructions.
Five microgram of outer membrane preparations were separated on a 4-15% TGX gel and analyzed by colloidal Coomassie staining (Pink et al., 2010).
Acknowledgments
This protocol was published in: de Vries et al. (2013). This study was financially supported by Vienna Spot of Excellence (VSOE) grant (ID337956).
References
de Vries, S. P., Burghout, P., Langereis, J. D., Zomer, A., Hermans, P. W. and Bootsma, H. J. (2013). Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions. Mol Microbiol 87(1): 14-29.
Pink, M., Verma, N., Rettenmeier, A. W. and Schmitz-Spanke, S. (2010). CBB staining protocol with higher sensitivity and mass spectrometric compatibility. Electrophoresis 31(4): 593-598.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Vries, S. P. D. and Bootsma, H. J. (2013). Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles. Bio-protocol 3(21): e957. DOI: 10.21769/BioProtoc.957.
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Category
Microbiology > Microbial proteomics > Membrane proteins
Systems Biology > Proteomics > Outer membrane proteins
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958 | https://bio-protocol.org/en/bpdetail?id=958&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Ki67 Immunofluorescence on Bovine Cell Lines
Justine Marsolier
SM Souhila Medjkane
MP Martine Perichon
JW Jonathan B. Weitzman
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.958 Views: 10261
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS Pathogens Apr 2013
Abstract
This is a rapid protocol to test the effects of drugs treatment on bovine cell replication using Ki67 staining. Ki67 is associated with cell proliferation and is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0).
Keywords: Ki67 Immunofluorescence Bovine lymphocytes
Materials and Reagents
Bovine cells (The TBL3 cell line was derived from in vitro infection of the spontaneous bovine-B lymphosarcoma cell line, BL3, with Hissar stock of T. annulata)
RPMI 1640 (Gibco)
Fetal calf serum (heat-inactivated)
Penicillin/streptomycin
Fibronectin (Sigma-Aldrich, catalog number: F1141 )
Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
SVF (PAA Laboratories GmbH, catalog number: A15-101 )
BSA (Sigma-Aldrich, catalog number: A2153 )
Mouse monoclonal anti-Ki67 (1:50) (Abcam, catalog number: ab10913-1 )
Cy2 AffinyPure anti-mouse IgG (1:5,000) (Jackson ImmunoResearch Laboratories, catalog number: 715-225-150 )
Tween 20 (Biosolve, catalog number: 2045 2335 )
ProLong Gold Antifade Reagent with Dapi (Life Technologies, Invitrogen™, catalog number: P-36931 )
DAPI
Equipment
Slides (Knittel Glass, catalog number: KN00010025787 )
37 °C, 5% CO2 cell culture incubator
24 wells plate
Fluorescent microscope (Leica Microsystems, model: Inverted 6000 )
Procedure
Bovine cells were cultured in RPMI 1640, supplemented with 10% heat-inactivated Fetal calf serum, 4 mM L-Glutamine, 25 mM HEPES, 10 μM β-mercaptoethanol and 100 μg/ml penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37 °C.
In 24 wells plate, slides were coated with PBS – Fibronectin (1/1,000), 2 h at 37 °C and wash twice with PBS.
Non adherent bovine cells (500,000 cells/well in 24 wells plate) were plated on Fibronectin coated slides 2 h at 37 °C, 5% CO2 and then fixed in 500 μl PBS 3.7% Formaldehyde for 15 min at room temperature.
Slides were rinsed in PBS (300 μl - 3 washes) and permeabilized with PBS 0.2% Triton X-100 for 5 min.
Slides were washed in PBS (300 μl - 3 washes) and then blocked for 30 min at room temperature with 250 μl PBS 1% SVF and 1% BSA to prevent non-specific staining.
The slides were incubated with Mouse monoclonal anti-Ki67 (1:50) in 100 μl PBS 1% SVF and 1% BSA at room temperature for 40 min.
After washing in PBS 0.2% Tween (300 μl - 3 washes), the slides were incubated with Cy2 AffinyPure anti-mouse IgG (1:5,000) in 100 μl PBS 1% SVF and 1% BSA for 30 min at room temperature, in the dark.
Slides were subsequently washed in PBS 0.2% Tween (300 μl - 3 washes), mounted on slides and coverslippedwith ProLong Gold Antifade Reagent with DAPI.
Images of immunofluorescence staining were photographed with a camera attached to a fluorescent microscope and percentage of Ki67 positive cells was calculated.
This staining was repeated for three independent biological replicates (Figure 1).
Figure 1. Ki67 staining of bovine cells
Acknowledgments
This protocol was adapted form James et al. (2005). The Weitzman laboratory was supported by Fondation de France (FdF #2102), Association pour le Recherche contre le Cancer (ARC Fixe #4975, ARC-Equipement #7990) and Association for International Cancer Research (AICR, #08-0111).
References
James, D., Levine, A. J., Besser, D. and Hemmati-Brivanlou, A. (2005). TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132(6): 1273-1282.
Marsolier, J., Pineau, S., Medjkane, S., Perichon, M., Yin, Q., Flemington, E., Weitzman, M. D. and Weitzman, J. B. (2013). OncomiR addiction is generated by a miR-155 feedback loop in Theileria-transformed leukocytes. PLoS Pathog 9(4): e1003222.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Marsolier, J., Medjkane, S., Perichon, M. and Weitzman, J. B. (2013). Ki67 Immunofluorescence on Bovine Cell Lines. Bio-protocol 3(21): e958. DOI: 10.21769/BioProtoc.958.
Marsolier, J., Pineau, S., Medjkane, S., Perichon, M., Yin, Q., Flemington, E., Weitzman, M. D. and Weitzman, J. B. (2013). OncomiR addiction is generated by a miR-155 feedback loop in Theileria-transformed leukocytes. PLoS Pathog 9(4): e1003222.
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Category
Cell Biology > Cell imaging > Fluorescence
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959 | https://bio-protocol.org/en/bpdetail?id=959&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Immunoplaque Assay (Influenza Virus)
Longping V. Tse
YZ Yueting Zhang
GW Gary R. Whittaker
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.959 Views: 24535
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Original Research Article:
The authors used this protocol in Journal of Virology Jun 2013
Abstract
Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious assay (TCID50) and genetic material quantification by Q-PCR provide a different way of viral quantification with their own shortcoming. In here, we modified the fluorescent focus assay and developed an antibody-based immunoplaque assay which provides a reliable and reproducible viral quantification independent of CPE. Our assay not only allows accurate determination of viral titer, but also provides information on viral kinetics, genetic stability and purity of the virus population.
Keywords: Influenza Plaque Assay Quantification
Materials and Reagents
MDCK cells
Please note that there are multiple variants of MDCK on American Type Culture Collection (ATCC). This protocol has been tested for MDCK (CCL-34), MDCK.1 (CRL-2935) and MDCK.2 (CRL-2936)
Recombinant A/California/04/2009 virus produced from 12 plasmids reverse genetic system (Generous gift from Professor Toru Takimoto from University of Rochester, New York, USA) (Bussey et al., 2010)
Trypsin/EDTA (Mediatech, Cellgro®, catalog number: 25-053-CI )
SeaPlaque Agarose (Lonza, catalog number: 50100 )
TPCK-Trypsin (Thermo Fisher Scientific, catalog number: 20233 )
PBS without Ca2+ and Mg2+ (Mediatech, Cellgro®, catalog number: 21-030-CV )
DPBS with Ca2+ and Mg2+ (Mediatech, Cellgro®, catalog number: 21-040-CV )
10% formalin (Sigma-Aldrich, catalog number: HT501128-4L )
TritonX-100 (Sigma-Aldrich, catalog number: T9284 )
5% goat serum in DPBS (Gibco, catalog number: 16210-072 )
Mouse anti-NP antibody from hybridoma H16-L10-4R5 (ATCC, catalog number: HB-65 )
Alkaline phosphatase conjugated Donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, catalog number: 715-056-150 )
Alexa Fluor 488 conjugated Goat anti-mouse antibody (Invitrogen, catalog number: A11001 )
BCIP/NBT substrate (Vector Laboratories, catalog number: SK-5400 )
DMEM (Mediatech, Cellgro®, catalog number: 10-017-CV )
DMEM powder (Mediatech, Cellgro®, catalog number: 50-003-PB )
Heat-inactivated fetal bovine serum (Mediatech, Cellgro®, catalog number: 26140079 )
HEPES (Mediatech, Cellgro®, catalog number: 25-060-CI )
Penicillin/Streptomycin (Mediatech, Cellgro®, 30-002-CI )
7.5% BSA Fraction V (Gibco, catalog number: 15260 )
RPMI 1640 powder (Mediatech, Cellgro®, catalog number: 50-020-PB )
Tris base (US Biological, catalog number: T8600 )
Sodium chloride (NaCl)
Magnesium chloride (MgCl2)
Sodium hydroxide (NaOH)
Complete DMEM (cDMEM) (see Recipes)
2x DMEM (see Recipes)
RPMI (see Recipes)
0.5% Triton X-100 in DPBS (see Recipes)
Alkaline phosphatase (AP) reaction buffer (see Recipes)
Equipment
Standard tissue culture equipment
12-well tissue culture plate
37 °C, 5% CO2 cell culture incubator
Inverted fluorescence microscope (Optional)
Rocker
Laboratory spatula
Heated stir plate
Vortex
Axiovert 200 microscope
Software
IPlab3.6.5 software
Procedure
Day 1
Each well on a 12-well plate is seeded with 5 x 105 MDCK cells suspended in 1 ml of cDMEM.
The plate is incubated in the 5% CO2 incubator at 37 °C overnight.
Day 2 (or until cells reach 95% - 100% confluence)
Viruses are serially diluted (10-fold) in RPMI, vortex each dilutions and keep on ice before use.
MDCK cells are rinsed with 1 ml of DPBS twice.
MDCK cells are inoculated with 500 μl of viruses prepared from step 3.
The plate is incubated in 5% CO2 incubator at 37 °C for 45 min.
Prepare SeaPlaque DMEM (pDMEM)
2x DMEM is warmed at 37 °C water bath and 2% SeqPlaque agarose is melted on a hot plate at about 45–50 °C with stirring (*The cap should be slightly released to prevent pressure buildup).
To obtain pDMEM, pre-warm 2x DMEM and 2% agarose are mixed in 1:1 ratio in a sterile container and sit at room temperature for 5–10 min to cool down.
TPCK-trypsin (final concentration: 1 μg/ml) is added to the pDMEM.
Viral inoculums are aspirated and MDCK cells are rinsed with 1 ml of DPBS once.
1.5 ml pDMEM + TPCK-trypsin from step 7-c is added to each well.
The plate is sat at room temperature (RT) for 10–15 min until pDMEM solidify.
The plate is incubated at 5% CO2 at 37 °C in the invert orientation (lid at the bottom) for 3 days.
Day 5 (or until visualization of plaque)
MDCK cells are fixed by adding 1.5 ml of 10% formalin directly on top of the agarose and incubated at RT for 2 h.
Extra formalin is aspirated and agarose from the well is removed and trashed carefully without disturbing the monolayer of cell using a laboratory spatula or equivalent tools.
Note: Experiment can be stopped after this step by adding PBS to cover the cells and store at 4 °C.
MDCK cells are permeabilized by 300 μl of 0.5% Triton-X100 in DPBS for 5 min on bench without agitation.
Cells are rinsed with 1 ml of PBS twice.
Cells are blocked by 300 μl of 5% goat serum in DPBS for 30 min at RT with agitation on rocker.
Cells are incubated with H16-L10-4R5 antibody (1:40) in 300 μl of 5% goat serum for 1 h at RT with agitation on a rocker.
Cells are rinsed with 1 ml of PBS twice.
Cells are incubated with alkaline phosphatase conjugated donkey anti-mouse (1:1,000) in 300 μl of 5% goat serum for 45 min at RT with agitation on a rocker.
Cells are rinsed with 1 ml of PBS twice and AP reaction buffer once.
Prepare the BCIP/NBT solution.
Added 2 drops of solution A, B and C into every 5 ml of AP reaction buffer.
Mix by inverting the tube for 4–6 times.
300 μl of BCIP/NBT solution is added to each well. The plate is put on a rocker in dark for 30 min or until the development of color.
The reaction is stopped by rinsing the plate with ddH2O.
The plate is air-dried at RT.
Count the number of plaques, check the plaque morphology and measure the size of plaques.
Alternative visualization method by fluorescence, fluorescent focus assay (FFA)
(Continuous from step 18)
19.1
Cells are incubated with goat anti-mouse Alexa Fluor 488 antibody (1:1,000) in 300 μl of 5% goat serum for 45 min at RT in dark.
20.1 Cells are rinsed with 1 ml of PBS twice.
21.1 Cells are covered with 500 μl of DPBS and store at 4 °C.
22.1
Check the plaque morphology and measure the size of plaques using an inverted fluorescence microscope (Figure 1).
Note: The protocol is optimized for influenza viruses. Modifications may be required for use in other viruses. For instance, factors required for multiple rounds of replication of the virus should be added in the pDMEM.
Figure 1. Diameter of Individual Plaque of A/California/04/2009/H1N1 Recombinatant Viruses (rCA0409). A. Quantification of the diameter (μm) of 100 individual plaques of two different CA0409 viruses, wild type (WT) and HA-S328Y mutants (HA328Y). Images are taken by Axiovert 200 microscope using a 5x objective and diameters of each plaque are measured by IPlab3.6.5 software. Statistics are done by one-tail Student’s t-test. B. Representative image of plaques of CA0409 viruses stained with Alexa Fluor 488, scale bar = 200 μm. Images are taken by Sensicam using IPlab 3.6.5 (Data not published) (Tse et al., 2013).
Recipes
cDMEM (filtered in 0.2 μm filtration unit)
DMEM
432.5 ml
Heat-inactivated fetal bovine serum
50 ml
1 M HEPES
12.5 ml
Penicillin/Streptomycin
5 ml
2x DMEM (filtered in 0.2 μm filtration unit)
DMEM powder
13.48 g
7.5% BSA Fraction V
26.7 ml
1 M HEPES
12.5 ml
Penicillin/Streptomycin
5 ml
ddH2O to 500 ml
RPMI (filtered in 0.2 μm filtration unit)
RPMI 1640 powder
5.2 g
7.5% BSA fraction V
13.3 ml
1 M HEPES
12.5 ml
Penicillin/Streptomycin
5 ml
ddH2O to 500 ml
0.5% Triton X-100 in DPBS
Triton X- 100
1 ml
DPBS
199 ml
AP reaction buffer
Tris
100 mM
NaCl
100 mM
MgCl2
5 mM
Adjust pH to 9.5 using NaOH
Acknowledgments
We thank Jean K. Millet, Tamar Friling, Alice M. Hamilton and all of the members of the Whittaker lab for helpful discussions. We also thank the Collins lab for helpful suggestions made throughout the study.
References
Bussey, K. A., Bousse, T. L., Desmet, E. A., Kim, B. and Takimoto, T. (2010). PB2 residue 271 plays a key role in enhanced polymerase activity of Influenza A viruses in mammalian host cells. J Virol 84(9): 4395-4406.
Tse, L. V., Marcano, V. C., Huang, W., Pocwierz, M. S. and Whittaker, G. R. (2013). Plasmin-mediated activation of pandemic H1N1 influenza virus hemagglutinin is independent of the viral neuraminidase. J Virol 87(9): 5161-5169.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Tse, L. V., Zhang, Y. and Whittaker, G. R. (2013). Immunoplaque Assay (Influenza Virus). Bio-protocol 3(21): e959. DOI: 10.21769/BioProtoc.959.
Download Citation in RIS Format
Category
Microbiology > Microbial cell biology > Cell isolation and culture
Cell Biology > Cell imaging > Fluorescence
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96 | https://bio-protocol.org/en/bpdetail?id=96&type=1 | # Bio-Protocol Content
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This is an In Press version of the protocol that has not yet been assigned to an issue.
Peer-reviewed
Making Yeast Competent Cells and Yeast Cell Transformation
Yongxian Lu
In Press
Published: Jul 20, 2011
DOI: 10.21769/BioProtoc.96 Views: 41019
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Abstract
This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells.
Materials and Reagents
Bacto-Yeast extract (Thermo Fisher Scientific)
Bacto-peptone (Thermo Fisher Scientific)
Glucose (dextrose) (Thermo Fisher Scientific)
Bacto-agar (Thermo Fisher Scientific)
Deionized H2O
Glycerol (Sigma-Aldrich)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich)
PEG 3350 (Sigma-Aldrich)
Lithium acetate dihydrate (LiAc) (Sigma-Aldrich)
Salmon sperm DNA (Life Technologies, Invitrogen™)
Yeast nitrogen base (YNB) (Sigma-Aldrich)
2-(N-morpholino) ethanesulfonic acid (MES)
ADE (Sigma-Aldrich)
Agar
YPAD plate & liquid medium (see Recipes)
Transformation solution (see Recipes)
YNB + MA plate (see Recipes)
Equipment
Water bath (VWR International)
Centrifuges (Eppendorf)
30 °C shaker and incubator (VWR International)
Standard petri dishes (VWR International)
1.5 ml centrifuge tubes (Eppendorf)
Procedure
Make yeast competent cells (Modified from Gietz & Schiestl, 2007)
Obtain yeast strains of interest and streak on YPAD plates. Let cells grow 2 d before inoculation.
1st Inoculation: Inoculate one colony into 25 ml YPAD liquid medium. Grow cells overnight at 30 °C with shaking speed around 200 rpm.
2nd inoculation. Transfer the 25 ml cell culture into 75 ml YPAD medium. Grow cells at 30 °C for 4 h.
Harvest cells by centrifugation at 3,000 x g for 5 min, wash cells with 0.5 volumes of sterile H2O.
Centrifuge again with 3,000 x g for 5 min.
Re-suspend cells in 0.01 volumes of sterile H2O, transfer to a suitable centrifuge tube and pellet at 3,000 x g for 5 min at 20 °C.
Re-suspend cell pellet in 0.01 volumes of filter sterilized frozen competent cell solution (5% v/v glycerol, 10% v/v DMSO).
Dispense 50 µl cells into 1.5 ml Eppendorf tubes.
Place the tubes into a box with Styrofoam or cardboard (slow freezing is essential for good survival rates).
Store the box in a -80 °C freezer (cells can be kept at this condition for up to one year).
Yeast cell transformation
If using competent cells stored at -80 °C, thaw cells at 37 °C water bath for 15-30 sec. if using freshly made competent cells, go to step 2).
Centrifuge at 13,000 x g for 2 min to remove the supernatant.
Make the transformation solution for the planned number of transformations plus one extra (negative control) (see the recipes).
Add the solution to the cell pellet, vortex to re-suspend the cells.
Incubate in a 42 °C water bath for 40 min.
Centrifuge at 13,000 x g for 30 sec and remove the supernatant.
Pipette 1 ml of sterile H2O into the transformation tube to re-suspend the pellet.
Plate 200 µl of the cell suspension onto the YNB + MA plate growth plate with your selection marker.
Incubate plates at 30 °C for 2~4 d.
Recipes
Make YPAD plate & liquid medium
1% Bacto-yeast extract - 10 g/L
2% Bacto-peptone -20 g/L
2% Glucose (Dextrose) - 20 g/L
If making YPD plates, add 20 g Bacto-agar.
Fill up to 1 L with deionized H2O.
NO pH adjustments.
Note: Autoclave agar and glucose separately, to avoid caramelization.
Transformation solution
PEG 3350 [50% (w/v)] 260 µl
1 M LiAc 36 µl
Salmon sperm DNA (10 mg/ml) 10 µl
Plasmid 3 µl
Sterile H2O 51 µl
Total 360 µl
YNB+ MA plate (100 ml)
YNB 0.67 g
20 mM MES 0.39 g
ADE 0.01 g
Agar 2 g
Glucose 2 g
References
Gietz, R. D. and Schiestl, R. H. (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 1-4.
Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M. A., Uozumi, N., Cheung, A. and Sze, H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.
Article Information
Copyright
© 2011 The Authors; exclusive licensee Bio-protocol LLC.
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Category
Cell Biology > Cell isolation and culture > Transformation
Microbiology > Microbial genetics > Transformation
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960 | https://bio-protocol.org/en/bpdetail?id=960&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells
Ningfei An
Yubin Kang
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.960 Views: 16576
Reviewed by: Lin Fang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Stem Cells Jun 2013
Abstract
Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during the S-phase of the cell cycle. As such, BrdU incorporation can be used to quantify the number of cells that are in S-phase in the time period during which BrdU is available. The following protocol describes an in vivo BrdU incorporation assay as a measure of cell proliferation in adult murine hematopioetic stem cells (HSCs). Specifically, BrdU incorporation was analyzed for long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, Lin-Sca-1+c-Kit+CD34+CD135+) population.
Materials and Reagents
Mouse
BrdU
RPMI1640
Fetal Bovine Serum (FBS)
Potassium bicarbonate
Ammonium chloride
EDTA
BSA
Lineage Cell Depletion Kit (including Biotin-Antibody Cocktail and Anti-Biotin MicroBeads) (Miltenyi Biotec, catalog number: 130-090-858 )
BD Cytofix/Cytoperm Buffer (Becton, Dickinson and Company, catalog number: 554714 )
BD Perm/Wash Buffer
DNase (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619 )
DPBS without calcium, magnesium (diluted from 10x DPBS) (Hyclone, catalog number: SH 30258.01 )
Antibodies:
PE-conjugated-Sca-I (Becton, Dickinson and Company, catalog number: 553336 )
APC-conjugated-c-Kit (Becton, Dickinson and Company, catalog number: 553356 )
PerCP-eFluor 710-conjugated-CD135 (eBioscience, catalog number: 46-1351-80 )
eFluor 450-conjugated CD34 (eBioscience, catalog number: 48-0341-80 )
BrdU-FITC (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619)
Buffer A (see Recipes)
Red blood cell lysis buffer (see Recipes)
Staining buffer (see Recipes)
Equipment
Small scissors and forceps
60 mm tissue culture dish
23 G needle
3 cc syringe
15 ml centrifuge tube
Cell strainer (Becton, Dickinson and Company, catalog number: 352340 )
Hemacytometer
MACS MS column (Miltenyi Biotec, catalog number: 130-042-201 )
MiniMASC separator (Miltenyi Biotec, catalog number: 130-042-102 )
Centrifuge
BD LSRFortessa Analytical Flow Cytometer
Procedure
Animal injection
i.p. Injection of BrdU (10 mg/ml) 100 μl to 8-12 weeks old mouse (50 μg/g BW, so 5 μl/g of BW), after 6 h, inject the second dose. 2 h post 2nd injection, euthanize the mice using CO2 method followed by cervical dislocation, obtain the bone marrow (BM) cells from 2 tibias and 2 femurs. Two injections ensure that BrdU can incorporate to DNA of both slow and quick turnover cells.
Obtain total bone marrow (BM) cells
Use small scissors and forceps, dissect out femurs and tibias from mice and place them in a 60 mm tissue culture dish containing 6 ml ice-cold RPMI1640 with 5% heat inactivated FBS. Use Kimwipe tissue to remove muscle and other tissues. Cut off both ends of each bone shaft in the dish.
Connect the end of the bone with 23 G needle on 3 cc syringe, flush out bone marrow with RPMI1640 with 5% heat inactivated FBS into the dish. Disaggregate bone marrow tissues by repeated aspirations using the same needle. Transfer the cell suspension to 15 ml centrifuge tube.
Spin down the cells at 350 x g for 5 min at room temperature, remove the supernatant, resuspend the cells in 1 ml of room temperature red blood cell lysis buffer and incubate at room temperature for 5 min, then add 5-10 ml of RPMI 1640 with 5% heat inactivated FBS.
Pass the cells through a cell strainer. Collect the flow through to a new tube. Take an aliquot and count the cells in a hemacytometer. Spin down at 350 x g for 5 min at room temperature. Remove the supernatant; the cell pellet should not contain any red color. Disaggregate the cell pellet and wash the cells one time with buffer A, spin down at 350 x g for 5 min at room temperature.
Lineage cell staining (follow the mouse Lineage Cell Depletion Kit)
Resuspend the total BM cells in buffer A (40 μl/107 cells).
Add Biotin-Antibody Cocktail (10 μl/107 cells) to stain the lineage differentiated cells. Cocktail of biotin-conjugated monoclonal antibodies contains anti-CD5, anti-CD45R(B220), anti-CD11b, anti-Gr-1(Ly-6G/C), anti-Neutrophil (7/4) and anti-Ter-119.
Mix well and incubate for 10 min at 4 °C.
Add additional buffer A in media (30 μl/107 cells) then add Anti-Biotin MicroBeads (20 μl/107 cells, provided in Lineage Cell Depletion Kit).
Mix well and incubate for 15 min at 4 °C.
Wash cell by adding 2 ml of buffer A. Centrifuge at 300 x g for 10 min at room temperature.
Remove the supernatant and resuspend the pellet in 0.5 ml of buffer A.
Lineage depleation
Place MACS MS column in MiniMASC separator.
Prepare column by rinsing with 0.5 ml buffer A.
Apply cell suspension onto the column. Allow the cells to pass through and collect flow through as Lin- fraction.
Wash column 3 times with buffer A (0.5 ml/each), wash each time once the column reservoir is empty.
Collect all the elute (Lin-) in one tube.
Count the Lin- cells, aliquot 1.5 x 106 cells to a new tube.
Add 2 times more staining buffer to the cell suspension.
Centrifuge the cells at 350 x g for 5 min.
Stain the Lin- cells with surface antigens:
Test Total (μl) Percp-eFluor 710 eFluor 450 PE APC Staining
buffer
CD135 CD34 Sca-I
CD117
(c-kit)
1 75 1 2 0.5 0.5 71
Stain each test sample per 1.5 x 106 cells/75 μl buffer, make antibody mix as following, for more samples, increase antibody amount and staining buffer proportionally.
Add 75 μl of antibody mix to the cell pellet. Incubate cells with antibodies for 15 minutes at room temperature (protected from light).
Wash one time with staining buffer. Spin down for 5 minutes at 350 x g, and discard the supernatant.
Fix and permeabilize the cells
Resuspend the cells in 100 μl of BD Cytofix/Cytoperm Buffer per tube.
Incubate the cells for 15 to 30 minutes at room temperature or on ice.
Wash the cells with 1 ml of 1x BD Perm/Wash Buffer (dilute the 10x buffer with deionized H2O). Centrifuge at 350 x g for 5 minutes at room temperature, and discard the supernatant.
Enhance the permeabilization:
Resuspend the cells in 100 μl of BD Cytoperm Permeabilization Buffer Plus per tube. This reagent is specially formulated for the BrdU Flow kit and is used as a staining enhancer and secondary permeabilization reagent.
Incubate the cells for 10 minutes on ice.
Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
Re-fix cells after secondary permeabilization:
Resuspend the cells in 100 μl of BD Cytofix/Cytoperm Buffer per tube.
Incubate the cells for 5 minutes at room temperature or on ice.
Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
Treat with DNase to expose incorporated BrdU
Resuspend the cells in 100 μl of diluted DNase (diluted to 300 μg/ml in DPBS) per tube, (i.e. 30 μg of DNase/106 cells).
Incubate cells for 1 hour at 37 °C.
Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 5c).
BrdU intracellular antigens staining
Make diluted BrdU antibody (1 μl to 50 μl/sample in BD Perm/Wash Buffer).
Incubate the cells for 20 minutes at room temperature.
Wash the cells in 1 ml of 1x BD Perm/Wash Buffer (as in step 4c).
Resuspend the cells in 0.3 ml of staining buffer and perform flow cytometry analysis. Samples can be stored overnight at 4 °C, protected from light, prior to analysis by flow cytometry.
Flow cytometry was performed on BD LSRFortessa Analytical Flow Cytometer with the following gating strategy (Figure 1).
Figure 1. Gating strategy to analyze BrdU incorporation in LT-HSCs. A. BM Lin- cells were labeled with PE-Scal-I and APC-c-Kit antibodies and analyzed by flow cytometry. B. Lin-/Scal-I+/c-Kit+ (LSK) cells were gated as showed in A and the LSK cells were further analyzed with eFluor 450-CD34 and PerCP-eFluor 710-CD135 staining. Long-term HSCs (LT-HSCs, shown as Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, shown as Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, shown as Lin-Sca-1+c-Kit+CD34+CD135+) were separated as indicated. C. BrdU incorporation was further analyzed for each cell population. Data shown are LT-HSCs population analyzed for BrdU staining.
Recipes
Buffer A
DPBS, pH 7.2 supplemented with 0.5% BSA and 2 mM EDTA
Red blood cell lysis buffer
155 mM potassium bicarbonate
10 mM Ammonium chloride
0.1 mM of EDTA, pH = 7.4
Staining buffer
DPBS, pH 7.2 supplememted with 0.5% BSA and 0.09% sodium azide
Acknowledgments
This protocol is adapted from An et al. (2013).
References
An, N., Lin, Y. W., Mahajan, S., Kellner, J. N., Wang, Y., Li, Z., Kraft, A. S. and Kang, Y. (2013). Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells. Stem Cells 31(6): 1202-1212.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
An, N. and Kang, Y. (2013). In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells. Bio-protocol 3(21): e960. DOI: 10.21769/BioProtoc.960.
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Category
Stem Cell > Adult stem cell > Hematopoietic stem cell
Cell Biology > Cell viability > Cell proliferation
Molecular Biology > DNA > DNA labeling
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961 | https://bio-protocol.org/en/bpdetail?id=961&type=0 | # Bio-Protocol Content
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Peer-reviewed
Mitochondrial Isolation and Purification from Mouse Spinal Cord
PP Philippe A. Parone
SC Sandrine Da Cruz
DC Don W. Cleveland
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.961 Views: 11432
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Mar 2013
Abstract
Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in the study of mitochondria following the discoveries that mitochondria are central to the process of programmed cell death and that mitochondrial dysfunctions are implicated in numerous diseases including a wide range of neurological disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis. In order to identify and study changes in mitochondrial function related to specific neurological conditions the mitochondria are often isolated from the compartment of the central nervous system most affected during disease. Here, we describe a protocol for the isolation of mitochondria from mouse spinal cord, a compartment of the central nervous system that is significantly affected in neuromuscular diseases such as amyotrophic lateral sclerosis. This method relies on differential centrifugation to separate the mitochondria from the other subcellular compartments.
Keywords: Mitochondria Isolation CNS ALS Neurodegeneration
Materials and Reagents
Isoflurane (Isosol) (Vedco, catalog number: 50201 )
Fatty acid (FA) free bovine serum albumin (Sigma-Aldrich, catalog number: 85041C )
cOmpleteTM ULTRA protease inhibitor tablets, EDTA-free (Roche, catalog number: 0589253001 )
OptiPrepTM density gradient medium (Sigma-Aldrich, catalog number: D1556 )
Bradford reagent (Bio-Rad, catalog number: 500-0001 )
Mannitol (Sigma-Aldrich, catalog number: M4125 )
Phosphate buffer saline (PBS) (see Recipes)
1 M Tris-HCl (see Recipes)
0.5 M EDTA (see Recipes)
Buffer M (see Recipes)
Buffer M + 0.45% FA free BSA + protease inhibitors (see Recipes)
Buffer M + protease inhibitors (see Recipes)
Equipment
10 ml disposable plastic syringe fitted (heat sealed) with a 200 μl pipette tip
Using a Bunsen burner, heat and partially melt the wide end of a 200 μl pipette tip. Before the plastic has time to harden quickly insert the melted end of the 200 μl pipette tip into the luer-lok of a disposable plastic 10 ml syringe. Let the assembly cool and check the tightness of the seal by running 10 ml of water at high pressure through the syringe/tip assembly.
Sharp heavy dissection duty scissors
Euthanasia jar
Balance and plastic weight boat
Tweezers
1 ml glass homogenizer with loose and tight pestles (Kimble Chase Kontes) (Fisher Scientific, catalog number: 885300-0001 )
Table top refrigerated centrifuge
Ultrafuge
Thinwall, Ultra-ClearTM 5 ml 13 x 51 mm ultrafuge tubes (Beckman Coulter, catalog number: 344057 )
SW55Ti swing-out rotor (Beckman Coulter, model: 342194 )
Pipettes
50 ml conical
Procedure
Preparation
Put the following items on ice:
Homogenizer + pestles (those should be placed in a 50 ml conical on ice).
50 ml of Buffer M + 0.45% BSA + cOmpleteTM.
50 ml of Buffer M + cOmpleteTM.
Transfer the following items to the cold room:
All the items placed on ice.
Pipettes
Pipette tips
Tweezers
Small bucket of water to rinse pestles
Swing out buckets for SW55Ti
UltraClear SW55Ti tubes
OptiPrep
Set up the animal dissection area with:
Euthanasia chamber with Isoflurane
Heavy duty scissors
10 ml syringe filled with PBS
Small plastic weigh trays placed on ice
Tweezers
The start of procedure
Figure 1. Procedure for purification of mitochondria from mouse spinal cord using differential centrifugation
Terminally anesthetize the mouse using isoflurane in the euthanasia chamber.
Cervicaly dislocate the animal, decapitate and section the spinal column at the iliac crest (just above the hips). Flush the spinal cord from the spinal column using the 10 ml syringe filled with PBS. Insert the tip at the caudal opening of the spinal column and eject about 5 ml of PBS. This should flush out the spinal cord from the rostral end of the severed spinal column. If a clean flushing of the spinal cord cannot be achieved, section the dorsal column below the front limbs and repeat the flushing procedure. In this case make sure to save the piece of spinal cord located within the small piece of spinal column resulting from the cut below the front-limbs. Place the spinal cord, after removing excess PBS, in the small plastic weigh trays placed on ice.
Weigh the spinal cord rapidly and place it back on ice.
Steps B5 to B18 are all performed in the cold room.
While working in the cold room, transfer the spinal cord with cold tweezers in the 1 ml glass homogenizer on ice. Add 10 volumes of buffer M + 0.45% FA free BSA + cOmpleteTM (e.g. if the spinal cord weighs 60 mg add 600 μl of buffer).
Gently homogenize the tissue on ice with 10-15 strokes (just enough to completely dissociate the tissue) using the cooled loose pestle. Avoid forming air bubbles.
Note: Generating bubbles during the homogenization process can lead to disruption of intracellular membranes and denaturing of proteins, both of which should be avoided to reduce damage to the mitochondria.
Gently Homogenize the tissue on ice with 15 strokes using the tight pestle. Avoid forming air bubbles.
Centrifuge at 1,000 x g for 5 min at 4 °C and transfer the supernatant (S1) to a fresh tube on ice.
Note:The 1,000 x g supernatant generated at this step is composed mostly of cytosol with heavy and light membranes. The pellet contains mostly unbroken cells and nuclei.
Resuspend the pellet (P1) in 10 volumes of buffer M + 0.45% FA free BSA + cOmpleteTM and gently homogenize 15x using the tight pestle. Avoid forming air bubbles.
Note:This step is used to further release cytosol and intracellular organelles from the remaining unbroken cells.
Centrifuge at 1,000 x g for 5 min at 4 °C and pool the resulting supernatant (S1) with the supernatant (S2) from the step 8.
Centrifuge the pooled supernatants at 1,000 x g for 5 min at 4 °C.
Note: This step ensures that any contaminating unbroken cells or nuclei are removed from the cytosol/intracellular membrane subcellular fraction.
Collect supernatant (S3) staying clear of the pellet and spin it at 12,000 x g for 10 min at 4 °C.
Prepare Beckman UltraClearTM tubes with 200 μl of OptiPrepTM and 600 μl of Buffer M + 0.45% FA free BSA + cOmpleteTM. Mix well and store on ice.
Discard the supernatant (S4) resulting from step B12 and gently (but thoroughly) resuspend the pellet (P4) using in 150 μl of buffer M + 0.45% FA free BSA + cOmpleteTM.
Note:The 12,000 x g supernatant generated at this step is composed mostly of cytosol with light membranes. The pellet contains mostly heavy intracellular membranes (mitochondria).
Once the pellet is completely resuspended transfer all of it (recording the final volume) into the Beckman UltraClearTM tubes prepared in step B13. Complement with buffer M + 0.45% FA free BSA + cOmpleteTM to make a final volume of 1 ml and mix gently (but thoroughly) with a 1 ml pipette.
Transfer the UltraClearTM tubes to the SW55Ti swing out buckets on ice and centrifuge the tubes in a SW55Ti rotor using a high speed centrifuge at 17,000 x g (equivalent to 13,400 rpm) at 4 °C for 17 minutes (remove breaks).
Note: The heavy membrane fraction is centrifuged in an OptiPrep gradient to remove most of the contaminants (for example myelin and endoplasmic reticulum) from the heavy membrane preparation. The UltraClearTM tubes can be reused if washed well after use (without detergent) and stored dry.
Gently remove the tubes from the buckets and place them on ice. Using gentle aspiration remove supernatant including the top white layer.
Gently resuspend the pellet (P5) in 1 ml of buffer M + cOmpleteTM (without completely dispersing it) and centrifuge at 12,000 x g for 10 minutes at 4 °C.
Note: After the OptiPrep gradient the heavy membranes are resuspended in buffer M without BSA so as to wash off the BSA from the previous steps to more accurately measure the protein concentration in step B20.
Resuspend the pellet (P6) in 10-20 μl of buffer M + cOmpleteTM by gently flicking the tube with your fingers and placing back on ice as quickly as possible. Make sure pellet is completely resuspended before performing the protein quantification.
Note: It is important to resuspend the heavy membranes at a high concentration since this reduces damage to the organelles. Resuspending the organelles by flicking also reduces organelle damage.
Estimate protein quantity using a Bradford assay on 1 μl of resuspended pellet. Once the protein concentration is determined, add a final concentration of 0.45% FA free BSA to the suspension using a 4.5% FA free BSA stock to avoid diluting the preparation.
Note: FA free BSA is added back to the heavy membrane preparation after protein quantification to allow for better preservation of mitochondrial activity by reducing uncoupling of the organelle. Ideally mitochondrial activity (oxygen consumption) should be measured on a fraction of the preparation to determine the quality of the organelle and the damage that may have been sustained during preparation.
Recipes
PBS (1 L)
Start with 800 ml of distilled water
Add 8 g of NaCl
Add 0.2 g of KCl
Add 1.44 g of Na2HPO4
Add 0.24 g of KH2PO4
Adjust the pH to 7.4 with HCl
Add distilled water to a total volume of 1 L
1 M Tris-HCl, pH 7.4 (500 ml)
Mix 60.57 g of Tris base with 400 ml of dH2O
When Tris base is dissolved pH to 7.4 using concentrated HCl
Complete to 500 ml with dH2O
Stored at 4 °C
0.5 M EDTA, pH 8.0 (500 ml)
Mix 73.06 g of EDTA with 400 ml of dH2O
Adjust pH to 8.0 using concentrated KOH
Complete to 500 ml with dH2O
Stored at 4 °C
Buffer M (500 ml)
Mix 19.1 g of mannitol into 400 ml of dH2O
Add 11.9 g of sucrose
Add 5 ml of 1 M Tris-HCl pH 7.4
Add 1 ml of 0.5 M EDTA pH 8.0
Complete to 500 ml with dH2O
Filter sterilize (0.22 μm)
Stored at 4 °C
Note: Before the start of the isolation procedure prepare Recipes 5 and 6.
Buffer M + 0.45% FA free BSA + protease inhibitors
50 ml Buffer M + 0.225 g FA free BSA + 1 tablet of cOmpleteTM protease inhibitors (stored at 4 °C)
Buffer M + protease inhibitors
50 ml Buffer M + 1 tablet of cOmpleteTM protease inhibitors (stored at 4 °C)
Acknowledgments
This protocol is adapted from Parone et al. (2013) and Vande Velde et al. (2008).
References
Parone, P. A., Da Cruz, S., Han, J. S., McAlonis-Downes, M., Vetto, A. P., Lee, S. K., Tseng, E. and Cleveland, D. W. (2013). Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci 33(11): 4657-4671.
Vande Velde, C., Miller, T. M., Cashman, N. R. and Cleveland, D. W. (2008). Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria. Proc Natl Acad Sci U S A 105(10): 4022-4027.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Parone, P. A., Cruz, S. D. and Cleveland, D. W. (2013). Mitochondrial Isolation and Purification from Mouse Spinal Cord. Bio-protocol 3(21): e961. DOI: 10.21769/BioProtoc.961.
Parone, P. A., Da Cruz, S., Han, J. S., McAlonis-Downes, M., Vetto, A. P., Lee, S. K., Tseng, E. and Cleveland, D. W. (2013). Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci 33(11): 4657-4671.
Download Citation in RIS Format
Category
Neuroscience > Cellular mechanisms > Mitochondria
Cell Biology > Organelle isolation > Mitochondria
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962 | https://bio-protocol.org/en/bpdetail?id=962&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)
Ralph T. Böttcher
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.962 Views: 11426
Reviewed by: Lin FangFanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Cell Biology Jun 2012
Abstract
Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular cell surface receptors, localize to different compartments within cells where they elicit distinct functions by interacting with specific proteins. Integrins represent a major family of cell surface receptors consisting of non-covalently associated α and β subunits that mediate the interaction of cells with their environment. However, integrins do not only localize to the cell surface but are also present in other compartments including the endoplasmic reticulum and endosomes where they engage with a distinct set of interacting partners or show distinct post-translational modifications. Standard immunoprecipitation of β1 integrins from a cell lysate without prior fractionation isolates β1 integrins from all compartments. In contrast, selective immunoprecipitation of cell surface β1 integrin allows enriching for the pool of β1 integrin on the cell surface thereby minimizing contaminations with β1 integrins from other subcellular compartments. To achieve this, living cells are incubated with a β1 integrin-specific antibody on ice to label cell surface β1 integrins prior to cell lysis and precipitation.
Keywords: Integrin Immunoprecipitation Selective Immunoprecipitation
Materials and Reagents
Mouse fibroblasts lacking β1 integrin (β1 -/-) or re-expressing wild-type β1 integrin (β1 wt)
Note: These cells are home-made immortalized mouse fibroblasts derived from floxed β1 parental cells. β1 -/- cells are used as negative control. However, the protocol can also be transferred to other mouse cell lines. When cells lacking β1 integrin are not available as negative control one has to include an unrelated antibody (see steps 2 and 4) to monitor for unspecific binding.
Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX-I (Gibco, catalog number: 31966-021 )
Fetal Bovine Serum (FBS) (PAA, catalog number: A15-101 )
PBS (Sigma-Aldrich, catalog number: P4417 )
0.5% Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 15400-054 )
Primaquine bisphosphate (Sigma-Aldrich, catalog number: 160393 )
Protein G sepharose (Protein G sepharose Fast Flow) (Sigma-Aldrich, catalog number: P3296 )
BCA Protein Assay (Thermo Fisher Scientific, catalog number: 23227 )
Triton X100
Tris-HCl
Na-deoxycholate
SDS
Glycerol
Bromphenol blue
Mercapthoethanol
Protease inhibitors (Complete Mini EDTA-free) (Roche, catalog number: 04 693 159 001 )
Phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 and 3) (Sigma-Aldrich, catalog numbers: P5726 and P0044 )
Talin-1 antibody (1:1,000 for western blotting) (Sigma-Aldrich, catalog number: T3287 )
SNX17 antibody (1:1,000 for western blotting) (Proteintech, catalog number: 10275-1-AP )
β1 integrin IP buffer (see Recipes)
2x Laemmli sample buffer (see Recipes)
Equipment
Cell scraper
10 cm cell culture dish
Centrifuge
37 °C, 5% CO2 cell culture incubator
26-G needle attached to 1-ml syringe
Heating block (Eppendorf Thermomixer compact or equivalent)
Procedure
Wash mouse fibroblasts expressing β1 integrin (β1 wt) and fibroblasts lacking β1 integrin (β1 -/-) with PBS, trypsinize and count cells using Glass slide with grids.
For both cell lines plate 2 x 106 cells per 10 cm dish and incubate in DMEM/10%FBS in the 5% CO2 incubator at 37 °C overnight.
Note: We used the cell line lacking β1 integrin as negative control. Alternatively, one can plate cells expressing β1 integrin for incubation with an unrelated control antibody (step 4).
Place dishes on ice and wash twice with ice-cold PBS (4 ml/dish).
To label cell surface β1 integrin, incubate the cells in 3 ml ice-cold DMEM/10%FBS containing the anti-β1 integrin antibody or an unrelated antibody as negative control.
Note: Anti-β1 integrin antibody: The antibody has to be directed against an epitope in the extracellular domain of integrin and has to recognize β1 integrin in its native conformation. We used a home-made antibody against mouse β1 integrin in a concentration of 1:1,500 (Bottcher et al., 2012). For other antibodies the amount has to be determined experimentally.
Control antibody: Should be derived from the same species as the anti-β1 integrin antibody and should be used in the same concentration.
Place dishes on rocker (approximately 7 see-saw movements per minute) at 4 °C and incubate for 60 minutes.
Place dishes on ice and wash twice with ice-cold PBS (4 ml/dish).
To selectively immunoprecipitate cell surface β1 integrin continue with step 9.
To immunoprecipitate β1 integrin from the endosomal compartment, incubate the cells in DMEM/10%FBS containing primaquine for 15 min at 37 °C.
Note: After antibody binding to cell surface β1 integrins on ice it is possible to induce β1 integrin endocytosis by incubating the cells at 37 °C. This enables the antibody-β1 integrin complexes to reach the endosomal compartment. β1 integrins are rapidly internalized and the addition of primaquine inhibits recycling of β1 integrin from endosomes back to the cell surface thereby enriching the amount of β1 integrin in endosomes. Depending on the cell type, 0.6 μM to 0.6 mM primaquine are used.
Place dishes on ice and wash once with ice-cold PBS (4 ml/dish).
Lyse cells in 1 ml β1 integrin IP buffer per 10 cm dish for 15 min on ice.
Scrape off cells and transfer the cell lysate into pre-cooled 1.5 ml reaction tubes.
Sonicate briefly or pass several times through a 26 gauge needle.
Spin cell lysate at 17,000 x g for 10 min at 4 °C.
Transfer the supernatant into fresh pre-cooled 1.5 ml reaction tubes.
Take out 60 μl for whole cell lysate sample and 5.0 μl to determine the protein concentration.
Take 30.0 μl Protein G sepharose slurry per 1 mg protein, wash three times with β1 integrin IP buffer and add equal amount of the cell lysis supernatant per sample (between 1.0-1.5 mg cell lysate was used per sample).
Incubate for 2 hours at 4 °C on a rocking platform or a rotator.
Spin the Eppendorf tube at 1,500 x g for 2 min at 4 °C. Remove the supernatant completely and wash the beads 3-5 times with 500 μl of β1 integrin IP buffer.
After the last wash, take off supernatant and elute proteins by heating to 90-100 °C for 7 minutes in 60 μl of 2x Laemmli sample buffer.
Spin at 10,000 x g for 30 sec, collect supernatant and load onto the gel. Supernatant samples can be collected and kept frozen at this point if the gel is to be run later.
Note: β1 integrin translation/processing is a tightly controlled step-wise process that starts with the synthesis of a 88 kDa polypeptide that undergoes sequential glycosylation in the ER (‘immature’ form 105 kDa) and in the Golgi giving rise to incompletely glycosylated β1 integrin subunit and a complete or ‘mature’ β1 subunit of around 125 kDa.
A successful immunoprecipitation of the cell-surface β1 integrins can be shown by western blotting with an antibody against β1 integrin (e.g. the antibody used for immunoprecipiation; dilution 1:10,000). The immature 105 kDa β1 integrin should be strongly reduced, ideally not detectable, after the immunoprecipitation. To further characterize the purity of your cell surface β1 integrins, the precipitate can be analyzed by western blotting for co-immunoprecipitated proteins such as talin-1 (interacts with β1 integrin at the plasma membrane; 1:1,000 for western blotting) or SNX17 (interacts with β1 integrin on endosomes; 1:1,000 for western blotting).
Recipes
β1 integrin IP buffer
50 mM Tris-HCl (pH 7.5)
150 mM NaCl
1% Triton X100
0.1% Na-deoxycholate
1 mM EDTA
Protease inhibitors (Complete Mini EDTA-free; 1 tablet for 10 ml of buffer)
Phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 and 3; 1:100 dilution from stock)
2x Laemmli sample buffer
120 mM Tris-HCl (pH 6.8)
4% SDS
20% glycerol
4 mM EDTA
0.001% bromphenol blue
2% mercapthoethanol
Acknowledgments
This protocol was adapted from a paper by Böttcher et al. (2012). We thank R. Fässler for critically reading the manuscript and continuous support. This work was funded by the Deutsche Forschungsgemeinschaft (SFB 914, project A05).
References
Bottcher, R. T., Stremmel, C., Meves, A., Meyer, H., Widmaier, M., Tseng, H. Y. and Fassler, R. (2012). Sorting nexin 17 prevents lysosomal degradation of β1 integrins by binding to the β1-integrin tail. Nat Cell Biol 14(6): 584-592.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Böttcher, R. T. (2013). β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation). Bio-protocol 3(21): e962. DOI: 10.21769/BioProtoc.962.
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Category
Biochemistry > Protein > Immunodetection > Immunoprecipitation
Cell Biology > Organelle isolation > Membrane
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963 | https://bio-protocol.org/en/bpdetail?id=963&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos
AM Almudena Martinez-Ferre
MN Maria Navarro-Garberí
CB Carlos Bueno
SM Salvador Martinez
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.963 Views: 12080
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Feb 2013
Abstract
Chick embryos are known to be a powerful system to test gene function due to the “in vivo” accessibility, short time for results retrieval and the possibility to perform a large number of experiments. Synthetic micro-beads soaked in morphogenetic signals or receptor inhibitors can be implanted in selective embryo regions at precise developmental stages activating or blocking different signaling pathways. Here, we describe the manipulation of Wnt signaling pathway using Dkk-1-soaked micro-beads, implanted in ovo in the anterior part of the developing neural tube of chicken embryos; specifically at the prospective zona limitans intrathalamica at stage HH10.
Keywords: Experimental embryology Neutral tube Wnt signaling regulation Microbeads implant Ni vivo experimentos
Materials and Reagents
Fertilized chick (Gallus gallus)
Recombinant mouse Dkk-1 (R&D Systems, catalog number: 1765-DK )
Heparin Acrylic beads (Sigma-Aldrich, catalog number: H5263 )
Bovine Serum Albumine (BSA) (Sigma-Aldrich, catalog number: A2153 )
Pelikan Drawing Ink Black
Silikon Peroxid (IDEX Health & Science, catalog number: SC0083ST )
Tungsten wire (0.380 mm, 10 FT) (World Precision Instruments, catalog number: TGW1510 )
Grid with concentric circles (NE42-21 mm) (Graticules LTD, Tonbridge Kent)
Penicillin-Streptomycin (10,000 U/ml) (Gibco, catalog number: 15140-122 )
NaCl (Sigma-Aldrich, catalog number: S-3014 )
KCl (Sigma-Aldrich, catalog number: P-9541 )
CaCl2.2H2O (Prolabo, catalog number: 22317297 )
NaH2PO4.2H2O (Scharlab, catalog number: SO0334 )
MgCl2.6H2O (Prolabo, catalog number: 27778293 )
D(+) glucose anhydre (Prolabo, catalog number: 24370294 )
10x Phosphate-buffer saline (PBS) (see Recipes)
4% Paraformaldehyde (PFA) from 20% PFA (see Recipes)
PBS/0.1% BSA (see Recipes)
Tyrodes buffer (see Recipes)
0.5% Bicarbonate (see Recipes)
0.5% Glucose (see Recipes)
Tyrodes supplemented (see Recipes)
Equipment
37 °C, 5% CO2 forced-air incubator (Novital, model: Covatutto 120-4V )
Dissecting microscope (Leica Microsystems, model: MS5 )
Butane burner (Butsir)
Glass micropipet (homemade)
Pasteur pipets (Deltalab, catalog number: 200001 )
Silicon tube (IDEX Health & Science, catalog number: SC0083ST )
Procedure
Fertilized chick (Gallus gallus) was incubated at 37 °C in a forced-air incubator. Chick embryos were developed until stage HH10 according to Hamburger and Hamilton (1951).
Glass micropipets were prepared using a Bunsen burner. One side of the micropipet was introduced into a silicon tube that was used to aspirate solution while picking up each bead (Figure 1A-D). Tungsten wire was joined to a Pasteur pipet to handle easily (Figure 1E).
Figure 1. Micropipets preparation. C-D. Tight part of Pasteur pipets was used to prepare micropipets. E. Wide part was used as a handle for the tungsten wire.
Use forceps to selective heparin acrylic beads based on their size. A grid inserted in one ocular of the operating microscope was used to select beads of ~40 μm. Afterwards, beads were rinsed in PBS and then soaked in a solution of 25 μg/ml of Dkk-1 protein in PBS/0.1% BSA or in PBS overnight (o/n) at 4 °C.
An opening in the shell was made with scissors. To counterstained the embryos in ovo, inject Indian ink (diluted 1:1 in Tyrode supplemented) under the blastoderm by a glass micropipet made with a Bunsen burner. The vitelline membrane that covers the embryo is slipped out with tungsten wire on the spot where microsurgery was to be performed (see Video 1).
Video 1. Counterstaining of chicken embryos in ovo
A grid with concentric circles was inserted in one ocular of the operating microscope. The working magnification used during microsurgery was 40x. This grid was positioned in relation to the embryo: the vertical axis followed the embryo ventral midline; the transversal axis was positioned by crossing the optic-diencephalic angle at both sides (Figure 2A) (Garcia-Lopez et al., 2004).
Afterwards, the soaked-beads were cut to obtain half-beads using forceps. Then they were rinsed in PBS several times and one soaked half-bead implanted in the right side of the neural tube of embryos (Figure 2B). For control experiments, beads were soaked in PBS in the same manner.
Figure 2. Bead-implanting procedure. A. Schematic representation of the grid positioned in relation to the embryo. B. Dkk-1 (black asterisk) or PBS-soaked beads were implanted into the prospective zona limitans intrathalamica of chick embryos at HH10. Abbreviations: Di, diencephalon; Mes, mesencephalon; Rh, rhombencephalon; Tel, telencephalon.
When the operation was completed, the opening in the eggshell was sealed with a piece of tape and incubated in horizontal position until the stage selected for fixation.
Recipes
10x PBS– phosphate-buffer saline
Mix 80 g of NaCl with 2 g of KCl, 14.4 g of Na2HPO4, 2.4 g of KH2PO4
Adjust pH to 7.4 with NaOH
Add dH2O to 1,000 ml
Filter sterilize (0.2 μm)
Stored at RT
20% PFA- paraformaldehyde
Mix 600 ml of preheated 1x PBS at 65 °C with 200 g of PFA
Add PBS to 1,000 ml
Adjust pH to 7.4 with NaOH
Filter sterilize (0.2 μm)
Stored at -20 °C
PBS/0.1% BSA
Mix 0.1 g of BSA with 100 ml of 1x PBS
Tyrodes buffer
4 g NaCl
0.1 g KCl
0.13 g CaCl2.2H2O
0.028 g NaH2PO4.2H2O
0.022 g MgCl2.6H2O
Add dH2O to 300 ml, sterilize and stored at 4 °C
0.5% Bicarbonate
0.5 g sodium hydrogen carbonate NaHCO3
Add dH2O to 100 ml, sterilize and stored at 4 °C
0.5% Glucose
0.5 g D(+) glucose anhydre
Add dH2O to 100 ml, sterilize and stored at 4 °C
Tyrodes supplemented
30 ml Tyrodes buffer
10 ml 0.5% Glucose
10 ml 0.5% Bicarbonate
500 μl 10,000 U/ml penicillin-streptomycin
Acknowledgments
This protocol was adapted from the previously published studies, Crossley et al. (1996) and Vieira and Martinez, (2005), and it was performed by Martinez-Ferre et al. (2013). This work was supported by EUCOMMTOOLS Contract 261492, Spanish Ministry of Science and Innovation Grant BFU-2008-00588, Spanish Ministry of Education and Science-Universitary Professor Formation Grant AP2009-3644, Consolider Grant CSD2007-00023, Institute of Health Carlos III, Spanish Cell Therapy Network and Research Center of Mental Health, General Council of Valencia (Prometeo 2009/028 and 11/2011/042), and the Alicia Koplowith Fondation. We thank M. Ródenas for technical assistance.
References
Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380(6569): 66-68.
Garcia-Lopez, R., Vieira, C., Echevarria, D. and Martinez, S. (2004). Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos. Dev Biol 268(2): 514-530.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J Morphol 88(1): 49-92.
Martinez-Ferre, A., Navarro-Garberi, M., Bueno, C. and Martinez, S. (2013). Wnt signal specifies the intrathalamic limit and its organizer properties by regulating Shh induction in the alar plate. J Neurosci 33(9): 3967-3980.
Vieira, C. and Martinez, S. (2005). Experimental study of MAP kinase phosphatase-3 (Mkp3) expression in the chick neural tube in relation to Fgf8 activity. Brain Res Brain Res Rev 49(2): 158-166.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Martinez-Ferre, A., Navarro-Garberí, M., Bueno, C. and Martinez, S. (2013). Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos. Bio-protocol 3(21): e963. DOI: 10.21769/BioProtoc.963.
Martinez-Ferre, A., Navarro-Garberi, M., Bueno, C. and Martinez, S. (2013). Wnt signal specifies the intrathalamic limit and its organizer properties by regulating Shh induction in the alar plate. J Neurosci 33(9): 3967-3980.
Download Citation in RIS Format
Category
Neuroscience > Development > Neuron
Developmental Biology > Morphogenesis
Cell Biology > Tissue analysis > Tissue staining
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964 | https://bio-protocol.org/en/bpdetail?id=964&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Visualization and Quantification of Actin Dynamics in Rice Protoplasts
Yurong Xie
Shanjin Huang
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.964 Views: 8873
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Plant Journal Mar 2013
Abstract
Direct visualization of the organization and dynamics of the actin cytoskeleton in rice cells is essential to understand its roles in regulating rice growth and development. Visualization of actin dynamics in protoplasts transformed with actin probe is a relatively quick and simple strategy, compared to the strategy of generating stable transgenic rice plants that harbor actin probe, which is time-consuming. Here is a protocol described in details regarding transforming rice protoplasts as well as visualizing and quantifying actin dynamics in rice protoplasts, which is based on the method previously reported (Shi et al., 2013).
Keywords: Rice protoplast Actin dynamics Filament severing frequency Actin depolymerization rate Filament elongation rate
Materials and Reagents
Rice protoplasts
Plasmid pUN1301-EGFP-ABD2-EGFP (Yang et al., 2011)
D-Mannitol (Sigma-Aldrich, catalog number: M1902 )
2-(N-Morpholino) ethanesulfonic acid (MES) (Merck KGaA, catalog number: 475893 )
Cellulase “Onozuka” RS (Yakult Pharmaceutical Industry, catalog number: 9012-54-8 )
Macerozyme R-10 (Yakult Pharmaceutical Industry, catalog number: 8032-75-1 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
β-mercaptoethanol (Merck KGaA, catalog number: 444203 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M2393 )
PEG4000 (Merck KGaA, catalog number: 817006 )
Murashige and Skoog basal salt mixture powder (MS) (Sigma-Aldrich, catalog number: M5524 )
Digestion buffer (see Recipes)
W5 buffer (see Recipes)
Mmg buffer (see Recipes)
Polyethylene glycol solution (see Recipes)
Equipment
35 μm nylon mesh
Spinning disk confocal microscope equipped with a Yokogawa CSU-X1 spinning disk head using a 512 x 512 Andor iXON electron-multiplying CCD camera (Andor)
Shaker
Centrifuge
Concave slide (diameter 15 mm) (Beyotime, catalog number: FSL011 )
Olympus BX61 inverted microscope equipped with a 100x 1.4 NA UPLSAPO (Universal Plan Super Apochromat Objective) objective
488 nm laser
525/50 nm band-pass filter
Software
Andor IQ2 software (available at http://www.andor.com)
Procedure
Sterilize rice seeds with 2.5% NaClO and 0.01% Tween-20 for 15 min and wash the seeds with sterilized water for five times, then sterilize the seeds with 2.5% NaClO for another 15 min and wash the seeds with sterilized water for five times. Finally put these seeds on sterilized filter paper and dry them.
Germinate and grow sterilized rice seeds above on half-strength MS medium at 28 °C for 14 d in darkness.
Cut the leaf sheaths of the etiolated seedlings into 0.5-1.0 mm length and immediately immerge into digestion buffer to digest the tissues in darkness at 28 °C for 4 h with gentle shaking (40-80 rpm). All steps below are operated at room temperature unless otherwise noted.
Add one volume of pre-cooling W5 buffer into above mixture and collect the protoplasts on ice by sifting out the undigested tissues with 35 μm nylon mesh.
Collect the protoplasts by a 5 min spinning of 300 x g at 4 °C and re-suspend the protoplasts in fresh cool W5 buffer.
Harvest the pellet and dilute the protoplasts into 2 x 106 cells/ml with Mmg buffer.
For transformation, 20 μg of plasmid pUN1301-EGFP-ABD2-EGFP was added into 100 μl protoplasts, followed by the addition of an equal volume of polyethylene glycol solution. The mixture was incubated at room temperature for 20 min.
Add 10 volume of W5 buffer and harvest the protoplasts with centrifugation at 300 x g for 5 min.
Re-suspend the pellet with fresh W5 buffer and incubate the protoplasts in darkness for 20 h.
Mount the protoplasts into a concave slide (diameter 15 mm) with a pipet and cover them with a coverslip. The dynamics of actin filaments were observed under an Olympus BX61 inverted microscope equipped with a 100x 1.4 NA UPLSAPO objective. Figure 1 shows the organization of actin filaments in a typical rice protoplast.
Figure 1. Organization of actin filaments in a typical rice protoplast. This protoplast is transformed with plasmid pUN1301-EGFP-ABD2-EGFP. Scale bar = 10 μm.
The images were collected using a spinning disk confocal microscope equipped with a Yokogawa CSU-X1 spinning disk head using a 512 x 512 Andor iXON electron-multiplying CCD camera. GFP was excited using a 488 nm laser, and fluorescence emission was collected using a 525/50 nm band-pass filter. Time-lapse Z-series images (with a step of 0.5 μm) were collected with a time interval of 100 msec using Andor IQ2, and the time interval for the whole Z-series collection was 5 sec. The Z-stack image is made into a movie to shown the actin filaments in a typical rice protoplast (see Video 1).
Video 1. A video of the Z-stack image displays the actin filaments in a typical rice protoplast harboring pUN1301-EGFP-ABD2-EGFP. Images were taken every 0.5 μm and were compressed into a 2.8 s-video. Scale bar = 10 μm.
The severing frequency of filament is quantified as the number of breaks, per unit filament length, per unit time (breaks/μm/s) and the depolymerization rate is calculated by the length of shortening filament within the given time interval (μm/s). Table 1 makes an example for one of our results.
Table 1. The severing frequency and depolymerization rate of filaments in rice protoplasts
Severing frequency (breaks/μm/s)
0.0165 ± 0.0080 (n = 15)
Depolymerization rate (μm/s) 0.0023 ± 6.58e-4 (n = 20)
Quantification of parameters associated with single actin dynamics in rice protoplasts harboring plasmid pUN1301-EGFP-ABD2-EGFP. More than 15 protoplasts were used to analyze. Values given are means± SE.
Recipes
Digestion buffer
0.6 M mannitol
10 mM MES pH 5.7
1.5% cellulose RS
0.75% macerozyme R10
0.1% BSA
1 mM CaCl2
5 mM β-mercaptoethanol
W5 buffer
154 mM NaCl
125 mM CaCl2
5 mM KCl
2 mM MES
Mmg buffer
0.6 M mannitol
15 mM MgCl2
4 mM MES
Polyethylene glycol solution
0.6 M mannitol
100 mM CaCl2
40% v/v PEG 4,000
Acknowledgments
This protocol was adapted from our previously published work (Shi et al., 2013). We thank Meng Shi and Shaojie Cui for helpful suggestions on rice protoplast preparation and transformation. The research in the Huang lab was supported by grants from Ministry of Science of Technology (2013CB945100) and National Natural Science Foundation of China (31125004).
References
Shi, M., Xie, Y., Zheng, Y., Wang, J., Su, Y., Yang, Q. and Huang, S. (2013). Oryza sativa actin-interacting protein 1 is required for rice growth by promoting actin turnover. Plant J 73(5): 747-760.
Yang, W., Ren, S., Zhang, X., Gao, M., Ye, S., Qi, Y., Zheng, Y., Wang, J., Zeng, L., Li, Q., Huang, S. and He, Z. (2011). BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development. Plant Cell 23(2): 661-680.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Xie, Y. and Huang, S. (2013). Visualization and Quantification of Actin Dynamics in Rice Protoplasts. Bio-protocol 3(21): e964. DOI: 10.21769/BioProtoc.964.
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Category
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Confocal microscopy
Plant Science > Plant cell biology > Cell structure
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965 | https://bio-protocol.org/en/bpdetail?id=965&type=0 | # Bio-Protocol Content
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Peer-reviewed
NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons
SZ Shu Zhang
YW Yu Tian Wang
Published: Vol 3, Iss 21, Nov 5, 2013
DOI: 10.21769/BioProtoc.965 Views: 11098
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience May 2013
Abstract
N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitotoxicity is thought to contribute to the pathogenesis of a large number of chronic neurodegenerative disorders (such as Alzheimer’s and Huntington’s diseases to mental illnesses) in addition to acute brain insults such as stroke and brain trauma. Understanding the mechanisms underlying NMDAR-mediated excitotoxicity may lead to development of novel therapeutics for treating neurological diseases. Stimulation of primary cultured neurons with excessive NMDA is widely used as an in vitro model for studying NMDAR-mediated excitotoxicity, which allows careful dissection of the detailed cellular mechanisms underlying excitotoxic neuronal death.
Lactate dehydrogenase (LDH) is a cytoplasmic enzyme which can convert NAD into NADH. LDH is released from cells into culture medium when the plasma membrane integrity is compromised. Therefore, the amount of released LDH represents the degree of cell death. In our current study, the extracellular LDH level was measured using an in vitro Toxicology Assay Kit obtained from Sigma-Aldrich. The basis of this LDH assay is: 1) LDH reduces NAD into NADH, 2) the resulting NADH is then utilized in the stoichiometric conversion of a tetrazolium dye, and 3) the resulting colored compound is measured by a spectrophotometric microplate reader at a wavelength of 490 nm. The cell death rate was expressed as a percentage (%) between the absorbance of treated group and that of control group.
Keywords: NMDAR Excitotoxicity Neuronal cultures LDH assay Rat
Materials and Reagents
Primary cultured neurons
NMDA (Sigma-Aldrich, catalog number: M3262-25MG )
Neurobasal medium (Life Technologies, Invitrogen™, catalog number: 21103 )
LDH Assay Substrate Solution
LDH Assay Dye Solution
LDH Assay Cofactor Preparation
In vitro Toxicology Assay Kit (Sigma-Aldrich, catalog number: TOX-7 )
Equipment
37 °C, 5% CO2 Cell culture incubator
96-well plate (Sigma-Aldrich, catalog number: SIAL0596-50EA )
Spectrometer for 96 well plate that can measure 490 nm and 690 nm (Molecular Devices, model: SpectraMax M2e Multi-Mode Microplate Reader)
Procedure
NMDA-induced Excitotoxicity
Prepare fresh NMDA 1,000x stock solution (25 mM) with fresh neurobasal medium. Old NMDA stock solution is less effective in inducing excitotoxicity, and higher dose might be required.
Immediately prior to NMDA treatment, half of the conditioned medium (old medium) is taken out and placed in the incubator to keep warm and pH balanced. The conditional medium taken out is saved for replacement of NMDA-containing medium after the excitotoxicity treatment. The reason to use conditional medium rather than fresh medium for replacement is that primary culture neurons are very sensitive to environment change, and using the conditional medium can minimize additional stimulation to neurons other than the NMDA treatment during the whole procedure.
NMDA is added directly to the culture medium to initiate excitotoxicity stimulation of neurons. The working concentration of NMDA is 25 μM. If the culture medium in the plate is 10 ml, the NMDA stock (25 mM) added will be 1/1,000 of 10 ml, which is 10 μl. Neurons are kept in the incubator during the treatment.
After 60 min incubation with NMDA, neurons are washed with fresh neural basal medium (warm and pH balanced) for once and then returned to the previously saved conditional medium.
Neurons are allowed to recover for different periods of time, ranging from 0 h to 24 h until further experiments.
Significant neuronal death could be observed after 6 h recovery and reaches its maximum level after 24 h.
Note: The conditioned medium is essential to minimize the stress for neurons.
Lactate dehydrogenase assay to detect cell death
Prepare the Lactate Dehydrogenase Assay Mixture immediately before performing the assay by mixing equal volume of LDH Assay Substrate Solution, LDH Assay Dye Solution and LDH Assay Cofactor Preparation according to Sigma manufacture protocol. Store the mixture on ice.
Remove cultures from incubator.
Transfer proximately 100 μl culture medium from each condition into a micro-centrifuge tube respectively and spin down the culture medium at 13,000 rpm for 1 min to deposit the cell debris.
Transfer 50 μl cultured medium from each condition into 96 well plate. Add 100 μl Lactate Dehydrogenase Assay Mixture to each sample. The volume of culture medium and Lactate Dehydrogenase Assay Mixture could be adjusted proportionally.
Cover the plate with Aluminum Foil to avoid light exposure.
Incubate at room temperature for several minutes to several hours depending on the concentration of LDH in the culture medium. For the first time user, reading the results every 20 min to determine the optimal end point is highly recommended. To obtain comparable results between different batches of cultures, a similar end point should be used. As the culture conditions (cell viability, cell density and medium volume) vary across different batches of neurons, it is sometimes hard to determine the end point using the length of reaction time. For example, in one batch of neurons, it may take 1 h to achieve a reading of 1.0 at 490 nm in the control group (non-treated group); while in another batch, it takes 1.5 h. Therefore, it’s highly recommended that the reaction should be read every 20 min during the whole LDH reaction, and only the data collected at the point when the reading of control group (e.g. 1.0 ± 0.1) is comparable to other batches should be used. As the reading is just arbitrary number, the data is only meaningful when comparing the treatment group with its control group.
Spectrophotometrically measure absorbance at a wavelength of 490 nm. Measure the background absorbance of 96 well plate at 690 nm. Subtract reading of 690 nm from that of 490 nm to obtain the final reading.
If plate reader is not available, samples could be transferred to appropriate sized cuvettes for spectrophotometric measurement.
Notes
The concentration of extracellular LDH depends on cell viability, cell density and medium volume. Ideally, cells density and medium volume in different culture wells should be exactly the same to avoid fluctuation of the results.
Some culture medium contains a significant level of LDH activity. In this case, blank medium should be measured and subtracted from the final results. Alternatively, serum-containing medium could be replaced with serum-free medium before any treatment.
Acknowledgments
This work was supported by the Canadian Institutes of Health Research (CIHR), CHDI Foundation, the Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111– 004), the National Research Council of Taiwan (NSC100-2632-B-039-001-MY3 and NSC 101-2320-B-039-059-), the National Natural Science Foundation of China 31040085 and 81271221, and Chongqing International Science and Technology Cooperation Foundation cstc201110003. Y.T.W. is a Howard Hughes Medical Institute International Scholar, and Heart and Stroke Foundation of British Columbia and Yukon Chair in Stroke Research. We thank Yuping Li, Dr. Henry Martin, Dr. Lidong Liu, and Dr. Jie Lu for technical support.
References
Decker, T. and Lohmann-Matthes, M. L. (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods 115(1): 61-69.
Legrand, C., Bour, J. M., Jacob, C., Capiaumont, J., Martial, A., Marc, A., Wudtke, M., Kretzmer, G., Demangel, C., Duval, D. and et al. (1992). Lactate dehydrogenase (LDH) activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium [corrected]. J Biotechnol 25(3): 231-243.
Liu, Y., Wong, T. P., Aarts, M., Rooyakkers, A., Liu, L., Lai, T. W., Wu, D. C., Lu, J., Tymianski, M., Craig, A. M. and Wang, Y. T. (2007). NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J Neurosci 27(11): 2846-2857.
Taghibiglou, C., Martin, H. G., Lai, T. W., Cho, T., Prasad, S., Kojic, L., Lu, J., Liu, Y., Lo, E., Zhang, S., Wu, J. Z., Li, Y. P., Wen, Y. H., Imm, J. H., Cynader, M. S. and Wang, Y. T. (2009). Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries. Nat Med 15(12): 1399-1406.
Zhang, S., Taghibiglou, C., Girling, K., Dong, Z., Lin, S. Z., Lee, W., Shyu, W. C. and Wang, Y. T. (2013). Critical role of increased PTEN nuclear translocation in excitotoxic and ischemic neuronal injuries. J Neurosci 33(18): 7997-8008.
Taghibiglou, C., Martin, H. G., Lai, T. W., Cho, T., Prasad, S., Kojic, L., Lu, J., Liu, Y., Lo, E., Zhang, S., Wu, J. Z., Li, Y. P., Wen, Y. H., Imm, J. H., Cynader, M. S. and Wang, Y. T. (2009). Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries. Nat Med 15(12): 1399-1406.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Zhang, S. and Wang, Y. T. (2013). NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons. Bio-protocol 3(21): e965. DOI: 10.21769/BioProtoc.965.
Zhang, S., Taghibiglou, C., Girling, K., Dong, Z., Lin, S. Z., Lee, W., Shyu, W. C. and Wang, Y. T. (2013). Critical role of increased PTEN nuclear translocation in excitotoxic and ischemic neuronal injuries. J Neurosci 33(18): 7997-8008.
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Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Cell viability > Cell death
Cell Biology > Cell staining > Protein
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966 | https://bio-protocol.org/en/bpdetail?id=966&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Mapping and Analysis of Illumina Reads for Transcriptome of Medicago Truncatula During the Early Organogenesis of the Nodule
AB Alexandre Boscari
AF Alberto Ferrarini
JG Jennifer del Giudice
LV Luca Venturini
MD Massimo Delledone
AP Alain Puppo
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.966 Views: 10352
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Plant Physiology Jan 2013
Abstract
Medicago truncatula serves as a model plant for legume genetics and genomics. We used RNA-Seq to characterize the transcriptome during the early organogenesis of the nodule and during its functioning. We generated approximately 135.5 million high-quality 36-bp reads, which were then aligned with the M. truncatula genome sequence (Mt3.0 version) and with sequences from a custom splice-junction database, for the detection of transcribed regions and splicing sites. Mapping and analysis of the reads conducted to the detection of 37,333 expressed transcription units (TUs), 1,670 had never been described before and were functionally annotated. We identified 7,595 new transcribed regions, mostly corresponding to 5’ and 3’ UTR extensions and new exons associated with 5,264 previously annotated genes. We also assessed the complexity in the nodulation transcriptome by performing a Cufflinks analysis to determine the frequency of the various alternatively spliced forms. Thus, we identified 23,164 different transcripts derived from 6,587 genes. Finally, we carried out a differential expression analysis, which provided a comprehensive view of transcriptional reprogramming during nodulation.
All Illumina sequence data have been deposited in the NCBI short-read archive, and Sanger-sequenced PCR products have been deposited in GenBank (SRA048731). Assembled contigs longer than 200 bp have been deposited at TSA (JR366937-JR375780). Coverage data are available at http://ddlab.sci.univr.it/cgi-bin/gbrowse/medicago/.
Keywords: Mapping illumina reads Medicago transcriptome Early organogenesis Nodule Medicago truncatula
Materials and Reagents
mRNA-Seq 8 sample prep kit (Illumina, catalog number: RS-100-0801 )
QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
RNeasy Plant Mini Kit (QIAGEN, catalog number: 74903 )
Standart agarose for electrophoresis (Sigma-Aldrich, catalog number: A9539 )
Equipment
Bioanalyzer Chip DNA 1000 series II (Agilent)
Gel electrophoresis apparatus
Illumina Genome Analyzer II (Illumina, model: SY-301-1201 )
Software
Bowtie (Langmead et al., 2009)
BEDTools suite (Quinlan and Hall, 2010)
TopHat (Trapnell et al., 2009)
Cufflinks (Roberts et al., 2011)
Velvet (Zerbino and Birney, 2008)
CAP3 (Huang and Madan, 1999)
GMAP (version 2012-04-21) (Wu and Watanabe, 2005)
Cistematic 2.5 (http://cistematic.caltech.edu/)
ERANGE software (3.1) (Mortazavi et al., 2008)
Medicago3.py: script to import Medicago annotation into cystematic (Boscari et al., 2012)
Gff2knowngene.pl: script to convert from General Feature Format (GFF) format to UCSC knowngene format (Zenoni et al., 2010)
CASAVA (Illumina)
GenomeStudio (Illumina)
Procedure
Poly(A) mRNA was isolated from the extracted RNA to prepare a nondirectional Illumina RNA-Seq library with mRNA-Seq 8 sample prep kit. We modified the gel extraction step by dissolving excised gel slices in QG buffer of QIAquick Gel Extraction Kit at room temperature to avoid under representation of AT-rich sequences.
Quality control and quantification of each library of 200 bp was performed with a Bioanalyzer Chip DNA 1000 series II.
36 to 44 bp sequences were generated on an Illumina genome analyzer II. A total of 135 million of reads were obtained for the different conditions.
M. truncatula Genome and the Splice Database Sequence alignments were generated with Bowtie (http://bowtie-bio.sourceforge.net).
Alignment of the reads was made on the Mt3.0 version of the M. truncatula genome sequence (www.medicago.org). For our analysis we allowed up to 2 mismatches, and sequences that matched with more than 10 different loci were discarded. Genome index is built with command “bowtie-build –f medicago.fasta genome” where medicago.fasta is the complete Mt3.0 fasta file. Reads are then aligned with command “bowtie -v 2 –m 10 –k 11 -S genome reads.fastq output.sam”, where reads.fastq are the raw sequencing reads of a sample, and the output.sam was processed using the software suite BedTools (http://code.google.com/p/bedtools/) in order to assign each read to an exon, intron, UTR, or intergenic region. Reads mapped onto external exons fell within a 3-kb catchment from both ends of a gene, promoting the investigation of putative undiscovered exons. Intergenic reads represented those sequence reads that fell outside this catchment. The program ERANGE defined potentially novel clusters of expression on the basis of their alignment; they were categorized as novel sections (exons/UTRs) of a known gene if they fell inside a radius of 3,000 bps from them (average gene density/2). The remaining expressed clusters were marked as potential new genes.
In order to identify potential new isoforms of known genes, we remapped all reads against the M. truncatula genome using TopHat (http://tophat.cbcb.umd.edu/) with a segment length of 16 due to the short length of our reads, and defined the new isoforms of known genes performing a Cufflinks (http://cufflinks.cbcb.umd.edu/) analysis on each sample, with standard parameters, followed by an analysis with Cuffcompare to merge transcripts identified on different samples. We used the latest genome sequence and annotations provided by the Medicago research community (Mt3.5, http://www.medicago.org/).
To identify novel transcribed regions, we used the reads which had not been mapped against the Mt3.0 sequence from every sample to assemble separately our contigs, using the Velvet program (http://www.ebi.ac.uk/~zerbino/velvet/), using a sensitive hash length of 29 for the reads with a length of 44 bps and of 21 for the rest. The contigs were subsequently clustered together using the software CAP3 (http://bioinformatics.ca/links_directory/tool/9319/cap3-sequence-assembly-program), with a minimum overlap of 90%, requiring an overlap identity of 80%. Contigs mapping against the reference genome with identity ≥ 90% and coverage ≥ 90% after BLAT alignment were discarded from further analysis. All the contigs were also mapped against the accompanying RNA-Seq data of the Mt3.5 version with GMAP (version 2012-04-21). The contigs, with an alignment coverage on the sequence length >= 90% and on the identity >= 90%, were merged together using the program mergeBed from the BEDTools suite (http://code.google.com/p/bedtools/).
The evaluation of gene expression was performed with the ERANGE software (3.1), available at http://woldlab.caltech.edu/RNA-Seq. ERANGE requires Cistematic 2.5 to execute RunStandardanalysis.sh. Therefore, a Python script (medicago3.py) was developed to import M. truncatula reference sequence (Mt3.0) and annotation in General Feature Format (GFF) into Cistematics Genomes sqlite database, and a Perl script (gff2knowngene.pl) was used to convert the GFF annotation file to the knowngene.txt file used by RunStandardanalysis.sh. ERANGE reports the number of mapped reads per kilobase of exon per million mapped reads, measuring the transcriptional activity for each gene. To obtain an accurate measure of gene expression not biased by reads mapping to splice junctions in genes with many introns, ERANGE considers both reads mapping to genome or to the custom splice junctions database. ERANGE was preferred over commercial packages such as CASAVA and GenomeStudio platform from Illumina because of its open nature. This allowed us to adapt and reuse code for our own analysis with greater flexibility than a comparable closed source commercial package.
Differential Gene Expression Statistic for RNA-Seq. ERANGE software computes the normalized gene locus expression level (named RPKM) by assigning reads to their site of origin and counting them. In the case of reads that match equally well to several sites, ERANGE assigns them proportionally to their most likely site(s) of origin (Mortazavi et al., 2008). The RPKM value for a given gene locus can be estimated as follows:
RPKM = N/(L * NTot) * 109
Where N = number of mapping reads at a given gene locus, L = estimated length (bp) of the gene locus, NTot = number of total mapping reads, and 109 is correspond to 1,000 bp transcript multiple 1 million reads. The null hypothesis of no differential gene expression for each gene was tested using the R package qvalue (Storey, 2002; Storey and Tibshirani, 2003; Dabney et al., 2010) on the R working environment. False Discovery Rates were calculated based on p-values obtained running a t test on the raw read counts using the basic R package stats.
Variance = (1/RPKM1) + (1/RPKM2)
Stat = (log(RPKM1/NTot1) - log(RPKM2/NTot2))/√(Variance)
p.value = (1-pnorm(abs(Stat))) * 2
The threshold value for the FDR was 0.001 and genes were first selected using this filter.
Differentially expressed genes were then filtered again based on a Fold Change (FC) > 2.
Acknowledgments
This protocol is adapted from Boscari et al. (2013).
References
Boscari, A., Del Giudice, J., Ferrarini, A., Venturini, L., Zaffini, A. L., Delledonne, M. and Puppo, A. (2013). Expression dynamics of the Medicago truncatula transcriptome during the symbiotic interaction with Sinorhizobium meliloti: which role for nitric oxide? Plant Physiol 161(1): 425-439.
Huang, X. and Madan, A. (1999). CAP3: A DNA sequence assembly program. Genome Res 9(9): 868-877.
Langmead, B., Trapnell, C., Pop, M. and Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3): R25.
Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. and Wold, B. (2008). Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5(7): 621-628.
Quinlan, A. R. and Hall, I. M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26(6): 841-842.
Roberts, A., Pimentel, H., Trapnell, C. and Pachter, L. (2011). Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics 27(17): 2325-2329.
Trapnell, C., Pachter, L. and Salzberg, S. L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9): 1105-1111.
Wu, T. D. and Watanabe, C. K. (2005). GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21(9): 1859-1875.
Zenoni, S., Ferrarini, A., Giacomelli, E., Xumerle, L., Fasoli, M., Malerba, G., Bellin, D., Pezzotti, M. and Delledonne, M. (2010). Characterization of transcriptional complexity during berry development in Vitis vinifera using RNA-Seq. Plant Physiol 152(4): 1787-1795.
Zerbino, D. R. and Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18(5): 821-829.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Boscari, A., Ferrarini, A., Giudice, J. D., Venturini, L., Delledone, M. and Puppo, A. (2013). Mapping and Analysis of Illumina Reads for Transcriptome of Medicago Truncatula During the Early Organogenesis of the Nodule. Bio-protocol 3(22): e966. DOI: 10.21769/BioProtoc.966.
Boscari, A., Del Giudice, J., Ferrarini, A., Venturini, L., Zaffini, A. L., Delledonne, M. and Puppo, A. (2013). Expression dynamics of the Medicago truncatula transcriptome during the symbiotic interaction with Sinorhizobium meliloti: which role for nitric oxide? Plant Physiol 161(1): 425-439.
Download Citation in RIS Format
Category
Plant Science > Plant molecular biology > RNA > RNA sequencing
Systems Biology > Transcriptomics > RNA-seq
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967 | https://bio-protocol.org/en/bpdetail?id=967&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay)
SP Saiprasad Goud Palusa
Anireddy S. N. Reddy
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.967 Views: 29301
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Plant Journal Apr 2013
Abstract
RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There are several hundreds of RBPs in plants, but the targets of most of them are unknown. A variety of experimental methods have been developed to identify targets of an RBP. These include RNA immunoprecipitation (RIP), UV cross-linking and immunoprecipitation (CLIP) and many variations of CLIP (e.g. PAR-CLIP, iCLIP). These approaches depend on immunoprecipitation of RNAs bound to a specific RBP using an antibody to that RBP. Electrophoretic mobility shift assay (EMSA), also called gel shift assay, has been used to analyze protein-nucleic acid interactions. It is a simple and powerful method to analyze protein-RNA/DNA interactions. In RNA EMSA, RNA-protein complexes are visualized by comparing the migration of RNA in the presence of a protein. Generally, in RNA EMSA a specific RNA sequence is used to analyze its interaction with a protein. In vitro transcribed 32P labeled or chemically synthesized RNA with a fluorescent tag is incubated with or without the protein of interest and the reaction mixture is then run on native polyacrylamide gel electrophoresis. RNA-Protein complexes migrate slowly as compared to free RNA, which can be visualized using an imaging system. In addition to test binding of an RBP to RNA, EMSA is also used to map the region in RNA and/or protein that is involved in interaction. Furthermore, the binding affinity can also be quantified using EMSA.
Materials and Reagents
Note: All work should be done in an RNase free environment, using only sterile, RNase free solutions and materials.
E. coli BL21 (DE3)-pLysS host cells harboring SR45 cDNA
In vitro transcribed RNA
Purified recombinant protein
Cap analogue (New England Biolabs, catalog number: S1404S )
SP6 polymerase (Fermentas, catalog number: EP0131 20 U/μl)
rNTPs
ATP (Fermentas, catalog number: R0441 )
CTP (Fermentas, catalog number: R0451 )
GTP (Fermentas, catalog number: R0461 )
UTP (Fermentas, catalog number: R0471 )
RNase Inhibitor (Life Technologies, Invitrogen™, catalog number: 10777019 )
32P-UTP- Uridine 5’ triphosphate (PerkinElmer, catalog number: BLU007C001 )
HEPES (Thermo Fischer Scientific, catalog number: AC172571000 )
KCl (Sigma-Aldrich, catalog number: P9541 )
MgCl2 (Mallinckrodt, catalog number: 5958-04 )
Glycerol (Thermo Fischer Scientific, catalog number: G331 )
Triton X-100 (Thermo Fischer Scientific, catalog number: BP151 )
Sodium dodecyl sulfate (SDS) (Mallinckrodt Baker, catalog number: 4095-02 )
NaCl (Thermo Fischer Scientific, catalog number: 7647-14-5 )
Na2HPO4 (Mallinckrodt, catalog number: 7917-04 )
KH2PO4 (Thermo Fischer Scientific, catalog number: 7778-77-0 )
Spermidine (Sigma-Aldrich, catalog number: 52626 )
Heparin sulfate (Sigma-Aldrich, catalog number: H4784 )
Tris base (AMRESCO, catalog number: 77-86-1 )
Acrylamide/bis-acrylamide (Bio-Rad Laboratories, catalog number: 16105B )
Phenol: chloroform (AMRESCO, catalog number: 0883 )
Ammonium persulfate (APS) (Gibco BRL®, catalog number: 5523UA )
TEMED (VWR International, catalog number: 97064-684 )
Bromophenol blue (Sigma-Aldrich, catalog number: 161-0404 )
Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
pGEM vector (Promega, catalog number: P2161 )
Isopropyl-β-D thiogalactopyranoside (IPTG) (Gold Bio, catalog number: I2481C5 )
Ampicillin (VWR International, catalog number: IB02040 )
S-Protein agarose beads (Merck KGaA, Novagen®, catalog number: 80031-014 )
Lysozyme (Sigma-Aldrich, catalog number: L6876 )
pET32c vector (Merck KGaA, Novagen®, catalog number: 69017-3 )
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
Boric acid (Mallinckrodt, catalog number: 2549-04 )
Ethylenediaminetetraacetic acid (EDTA) (Thermo Fischer Scientific, catalog number: S311-500 )
Ammonium acetate (Thermo Fischer Scientific, catalog number: A637-500 )
Bovine serum albumin (BSA)
LB medium
5x Gel shift buffer (see Recipes)
Lysis buffer (see Recipes)
10x TBE (see Recipes)
Gel Mix (see Recipes)
5% Denature Gel (see Recipes)
5% Non-Denaturing Gel (see Recipes)
TNS Solution (see Recipes)
RNA loading dye for Urea gel (see Recipes)
6x loading buffer for Non-Urea gel (see Recipes)
Binding/wash buffers (see Recipes)
Phosphate buffer (see Recipes)
Citrate buffer (see Recipes)
Equipment
1.5 ml microcentrifuge tube (BioExpress, catalog number: C-3262 )
Saran wrap
Whatman filter paper
Water bath (VWR International, model: 1225 PC )
Electrophoresis equipment with power supply (Thermo Fisher Scientific, model: B2 easy cost )
Gel drying apparatus (Savant System LLC, model: SGD 2000 )
Phosphorimager (Molecular Dynamics, model: Storm 840 )
Phosphorimager screen (Molecular Dynamics, model: 1000004864 )
Sonicator (SP Scientific, model: 274480 )
0.45 micron syringe filter (Life Science Products, catalog number: 25CSO80AS )
Centrifuge (Eppendorf, model: 022620401 )
Vertical gel apparatus (Gibco BRL®, model: V15.17 )
Liquid scintillation counter
Orbital shaker
Procedure
Note: We routinely produce RNA probes by in vitro transcription using a linearized pGEM clone that contains the DNA corresponding to RNA of interest. pGEM vector has T7 and SP6 promoters. Depending on the orientation of insert DNA either T7 polymerase or SP6 polymerase is used in in vitro transcription to generate RNA probe.
Preparation of RNA using in vitro transcription system
Prepare the following reaction in a 1.5 ml of microcentrifuge tube. 1 μl DNA template (~ 1 μg of linearized plasmid DNA), 1 μl 10x RNA polymerase buffer, 1 μl rNTPs (5 mM ATP, CTP, 0.5 mM GTP, UTP), 1 μl cap analog (5 mM), 4.5 μl (45 μCi): a-32P-UTP (800 Ci/mmol; 10 mCi/ml), 0.5 μl RNase Inhibitor, and 1 μl SP6 polymerase a total of 10 μl of reaction.
Incubate 37 °C, for 3 to 6 hours (Note 1).
Add 90 μl dH2O to 10 μl total 100 μl.
Add equal volume of (100 μl) of phenol/chloroform, mix by vortexing.
Spin at 16,000 x g for 5 min.
Transfer upper layer into a new Eppendorf (~100 μl).
Add 33 μl 10 M ammonium acetate and 250 μl 100% ethanol to the 100 μl.
Incubate at -80 °C for 10 min.
Spin at 16,000 x g for 10 min.
Wash the pellet with 500 μl of 80% ethanol.
Air dry the pellet; resuspend in 10 μl RNA loading dye.
Heat 1 min at 90 °C.
Load on 5% denaturing acrylamide gel and run for one to two hours depending on the size of RNA (Note 2).
Disassemble the gel chamber and dismantle the gel, leaving it mounted on one plate. To facilitate the alignment of the gel to the X-ray film in stepA16 below, cut one corner of the gel.
Wrap the gel with Saran wrap to avoid contamination.
Expose the gel to an X-ray film for 2-5 min and develop the film to visualize radioactive bands.
Align the gel on top of the X-ray film and locate the region in the gel corresponding to the radioactive band on the film.
Excise the gel corresponding to the band on the X-ray film (see Figure 1 and Notes 3 & 4).
Figure 1. In vitro transcribed RNA used in Figure 2a and 2b. After exposing the gel to an X-ray film, identified the right size of RNA product, excised the gel corresponding to the band and used to extract RNA.
Transfer the excised band to 400 μl of TNS solution in an Eppendorf tube and incubate overnight at room temperature.
Next day, take out TNS solution, which contains radiolabelled RNA and add equal volume of phenol/chloroform, mix by vortexing.
Spin at 16,000 x g for 10 min.
Wash the pellet with 500 μl of 80% ethanol.
Dry the pellet; resuspend the RNA pellet in 20 μl H2O.
Measure radioactivity in1 μl of RNA using liquid scintillation counter and use this number to calculate concentration of labeled “U” residues in RNA probe (Note 5).
Dilute RNA probe with RNase free water to 50 to100 K cpm/μl for use in EMSA assays.
Preparation and purification of recombinant protein
For EMSA analysis apart from RNA a purified protein of interest is needed. One can generate this protein by cloning the cDNA into a bacterial expression vector. Here we expressed SR45 in E.coli as an S.tag fusion and purified it using S-protein agarose beads.
Grow E. coli BL21 (DE3)-pLysS host cells harboring SR45 cDNA in LB medium containing ampicillin at 37 °C for overnight. Next day inoculate 1 ml of overnight culture into 100 ml of LB medium containing appropriate antibiotic, then grow culture until OD600 reaches ~0.6 (it will take approximately 2 h).
Induce expression of recombinant protein by adding IPTG to a final concentration of 0.5 mM and allow the bacterial culture to grow for an additional 4 h at 30 °C.
Harvest bacterial cells by centrifugation at 2,350 x g for 10 min at 4 °C.
Discard the supernatant and resuspend the cell pellet in 1/10 culture volume (5 ml) of 50 mM Tris-HCl pH 8.0, 2 mM EDTA.
Add lysozyme to a concentration of 100 μg/ml; use a 10 mg/ml stock freshly prepared in 50 mM Tris-HCl pH 8.0, 2 mM EDTA. Then add 1/10 volume (0.5 ml) of 1% Triton X-100. Incubate at 30 °C for 15 min.
Place the tube in an ice bath and sonicate 4 times (at 3.5 setting) for 15 sec/each time.
Centrifuge at 12,000 x g for 15 min at 4 °C. The supernatant contains soluble proteins.
Filter the supernatant through a 0.45 micron membrane.
Add 100 μl of binding/wash buffer to 100 μl of S-protein Agarose slurry in an Eppendorf tube and mix it gently, then add 1 ml of soluble proteins from step 8 (Note 6).
Mix and incubate at room temperature on an orbital shaker for 30 min. (Do not shake vigorously as this may denature protein).
Centrifuge at 500 x g for 10 min at 4 °C and carefully decant supernatant and wash the beads with five times, 1 ml each time, with binding/wash buffer.
Resuspend the washed beads containing the bound protein in 150 μl of the 0.2 M citrate buffer, pH 2.
Incubate for 10 min at room temperature, mix gently every few minutes.
Neutralized by adding 8 μl 2 M Tris-base (pH 10.4).
The eluted proteins were dialyzed against phosphate buffer (Note 7).
Electrophoretic Mobility Shift Assay (EMSA)
Before setting up the reaction, prepare 5% non-denaturing (native) gel, and pre-run the gel at 200 V using 1x TBE buffer for 10 to 15 minutes.
Prepare the following reaction mixture (final volume 14 μl) in a 1.5 ml of microcentrifuge tube. 1.5 μl of 1 mM spermidine (100 mM), 3 μl of 5x gel shift buffer, 0.5 μl of RNase Inhibitor, 1 μl of radiolabel RNA (50-100 K cpm), x μl lysis buffer (depending on protein volume) and x μl of protein of interest (Note 8).
Incubate at 30 °C for 5 min.
Add 1 μl of Heparin Sulfate (50 mg/ml) (Note 9).
Transfer the reactions to ice for 5 min.
Add 3 μl 6x urea free loading buffer.
Load the reaction products on 5% non-denaturing gel and run at 200 V for 2 to 3 h or as long as necessary for good separation (normally we will run 2 h for RNA probe that is about 200 nt) (Note 10).
Transfer the gel to Whatman filter paper, cover with Saran wrap, and dry with a gel-drying apparatus.
The dried gel should be exposed to the phosphorimaging screen for 2 h to overnight.
Free RNA and RNA – Protein complex(es) are visualized by using phosphorimager (see Figure 2a and 2b).
Figure 2. RNA EMSA. A. EMSA with an RNA probe using a purified recombinant protein. Lane 1, free probe; lanes 2-5, increasing concentration of recombinant protein. B. Binding of recombinant protein to RNA probe is competed by cold RNA. Lane 1, free probe; lane 2, probe + recombinant protein; lanes 3-7, as lane 2, with increasing concentration of cold RNA. Arrows indicate free probe, and the RNA-protein complex is indicated by arrowheads.
Competition with unlabeled RNA
After confirming the binding of a protein with a specific RNA target using EMSA, the specificity of interaction can be tested by competition studies in the presence of excess amount of same RNA that is not labeled. Increasing amount of competitor RNA (up to 100x of labeled RNA) is added to the binding reaction mixture and the ability of the cold competitor RNA to disrupt the complex is determined as above using native gel electrophoresis (see Figure 2b). Unlabeled competitor RNAs is generated in the same manner as above (see A section), except that the amount of cold UTP is increased to 5 mM and the amount of radioactive UTP is reduced to 0.045 μCi.
Notes
Three hours of incubation yields good amount of RNA.
For ~100 base long RNA probe run the gel for 1 h; for 200 to 300 nts long probe run the gel for two to three hours.
It is important to run in vitro transcribed RNA on a denaturing gel to confirm that the right size product is generated. The correct size band should then be excised and purified.
Sometimes RNA templates yield two or more RNA products due to its secondary structure. If this happens, excise only the correct size band for purification.
Concentration of labeled “U” in RNA is calculated using the specific activity of radiolabeled UTP, final concentration of UTP (radiolabeled UTP plus cold UTP) in the reaction and the number of “U” residues in the template. (fmoles of RNA = fmoles of UTP in the RNA probe/number of "U" residues in the labeled RNA).
Since the amount of protein in soluble fraction varies depending on the clone and expression level, it is necessary to optimize the ratio between agarose beads and the protein.
The recombinant protein should be purified to near homogeneity and protein concentration should be determined. It is not advisable to use crude extract from bacteria expressing recombinant protein.
Always use a known concentration of RNA probe and increasing concentration of protein.
Heparin sulfate reduces non-specific binding of RNA probe to proteins and eliminates background.
Running time of native gels is dependent on the RNA and protein complex, it must be optimized for each RNA and protein. For longer run times, gels must be run at 4 °C temperature.
Smaller size RNA probes (from 50 to 200 nts) work well in RNA EMSA.
If some RNA/protein complex stays in the well, add BSA to minimize this effect.
The concentration of RNA and protein should be optimized for each RNA-protein complex. Approximately 100,000 cpm of RNA and 500 ng of protein is a good starting point for binding studies.
Recipes
Note: All buffers should be prepared with RNase free water.
5x Gel shift buffer
70 mM HEPES pH 7.9
450 mM KCl
11 mM MgCl2
28% Glycerol
Lysis buffer
50 mM HEPES pH 7.9
150 mM KCl
1 mM MgCl2
1% Triton X–100
10% Glycerol
10x TBE
108 grams of Tris base
55 grams of boric acid
9.3 grams of EDTA dissolved in water
Made up to one liter and autoclaved
Use 1x as running buffer in running both denaturing and non-denaturing (Native) gels
Gel Mix (500 ml)
240 grams of Urea
50 ml of 10x TBE
200 ml H2O
Gel mix is stable for at least two to three months at room temperature
5% Denature Gel (30 ml)
5 ml of 30% Acrylamide/bisacrylamide
25 ml gel mix
300 μl 10% APS
30 μl TEMED
5% Non-Denaturing Gel
6.75 ml of 40% Acrylamide/bisacrylamide (38:2)
4.5 ml of 10x TBE
300 μl of 10% APS
30 μl of TEMED
33.6 ml of H2O
TNS Solution
25 mM Tris-HCl pH 7.5
400 mM NaCl
0.1% SDS
RNA loading dye for Urea gel
20 mM Tris-HCl pH 7.6
8 M Urea
1 mM EDTA
0.05% Xylene cyanol
0.05% Bromophenolo blue
6x loading buffer for Non-Urea gel
30% Glycerol
0.3% Bromophenol Blue
0.3% Xylene Cyanol
Binding/wash buffers
20 mM Tris-HCl, pH 7.5
150 mM NaCl
0.1% Triton X-100
1x protease inhibitors
Phosphate buffer
10 mM Na2HPO4
2 mM KH2PO4
2.7 mM KCl
137 NaCl pH 7.4
Citrate Buffer
2 M citric acid
Adjust pH to 2 with 10 M KOH and dilute to 0.2 M
Acknowledgments
This protocol was adapted from Thomas et al. (2012). This work was supported by a grant from the US National Science Foundation.
References
Day, I. S., Golovkin, M., Palusa, S. G., Link, A., Ali, G. S., Thomas, J., Richardson, D. N. and Reddy, A. S. (2012). Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing. Plant J 71(6): 936-947.
Golovkin, M. and Reddy, A. S. (1999). An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein. J Biol Chem 274(51): 36428-36438.
Palusa, S. G. and Wilusz, J. (2013). Approaches for the Identification and Characterization of RNA-Protein Interactions. Biophysical approaches to translational control of gene expression, Biophysics for the Life Sciences, J. D. Dinman (eds). Springer: 199-212.
Ryder, S. P., Recht, M. I. and Williamson, J. R. (2008). Quantitative analysis of protein-RNA interactions by gel mobility shift. Methods Mol Biol 488: 99-115.
Thomas, J., Palusa, S. G., Prasad, K. V., Ali, G. S., Surabhi, G. K., Ben-Hur, A., Abdel-Ghany, S. E. and Reddy, A. S. (2012). Identification of an intronic splicing regulatory element involved in auto-regulation of alternative splicing of SCL33 pre-mRNA. Plant J 72(6):935-946.
Wilusz, J. and Shenk, T. (1988). A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell 52(2): 221-228.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Palusa, S. G. and Reddy, A. S. N. (2013). Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay). Bio-protocol 3(22): e967. DOI: 10.21769/BioProtoc.967.
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Plant Science > Plant biochemistry > Protein > Interaction
Plant Science > Plant molecular biology > RNA > RNA-protein interaction
Molecular Biology > RNA > RNA-protein interaction
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968 | https://bio-protocol.org/en/bpdetail?id=968&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Gastric Aspiration Models
Bruce A. Davidson
RA Ravi Alluri
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.968 Views: 19081
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Original Research Article:
The authors used this protocol in The Journal of Immunology Feb 2013
Abstract
The procedures described below are for producing gastric aspiration pneumonitis in mice with alterations for rats and rabbits described parenthetically. We use 4 different injury vehicles delivered intratracheally to investigate the inflammatory responses to gastric aspiration:
1) Normal saline (NS) as the injury vehicle control
2) NS + HCl, pH = 1.25 (acid)
3) NS + gastric particles, pH ≈ 5.3 (part.)
4) NS + gastric particles + HCl, pH = 1.25 (acid + part.)
The volume, pH, and gastric particle concentration all affect the resulting lung injury. In mice, we generally use an injury volume of 3.6 ml/kg (rat: 1.2 ml/kg, rabbit: 2.4 ml/kg), an injury pH (for the acid-containing vehicles) of 1.25, and a gastric particulate concentration (in the particulate-containing vehicles) of 10 mg/ml (rat: 40 mg/ml). In our hands this results in a maximal, non-lethal lung injury with ≤ 10% mortality for the most injurious vehicle (i.e., acid + part.) The maximum tolerable particulate concentration needs to be determined empirically for any new strains to be used, especially in genetically-altered mice, because an altered inflammatory response may have detrimental affects on mortality.
We have extensive experience utilizing these procedures in the outbred strain, CD-1, as well as many genetically-altered inbred stains on the C57BL/6 background. Choice of strain should be carefully considered, especially in terms of strain-specific immune bias, to assure proper data interpretation. The size of the mouse should be ≥ 20 g at the time of injury. Smaller mice can be attempted, if necessary, but the surgical manipulation becomes increasingly more difficult and the surgery survival rate decreases substantially. There are no size or strain constraints for rat and rabbit models, but we generally use Long-Evans rats at 250-300 g and New Zealand White rats at ≈ 2 kg at the time of initial injury.
Keywords: Pneumonitis Acute lung injury (ALI) Acute respiratory distress syndrome (ARDS) Inflammation Rodent models
Materials and Reagents
Isoflurane
Topical antiseptic microbicide prep solution (e.g. Medline Industries, catalog number: MDS093906 )
0.5% bupivicaine
Hank’s Balanced Salt Solution (HBSS) with Ca2+, Mg2+ (e.g. Life Technologies, catalog number: 14025 )
HBSS without Ca2+, Mg2+ (e.g. Life Technologies, catalog number: 14175 )
Liquid nitrogen
Bovine serum albumin (BSA)
Cytospin filter cards (e.g. Thermo Fisher Scientific, catalog number: 5991022 )
Diff Quik solutions kit (Fisher Scientific, catalog number: NC9943455 )
Cytoseal 60 (VWR International, catalog number: 48212-154 )
100x protease inhibitor cocktail (e.g. Calbiochem®, catalog number: 80053-844 )
Bupivacaine
100 mg/ml mouse (rat) gastric particles (see Recipes)
Acid injury solution (acid) (see Recipes)
Gastric particles injury solution (part.) (see Recipes)
Acid + particles injury solution (acid + part.) (see Recipes)
Phosphate buffered saline (PBS), pH 7.2 (see Recipes)
Ammonium chloride lysis buffer (see Recipes)
Lung homogenate buffer (see Recipes)
50 mM potassium phosphate buffer, pH = 6.0 (see Recipes)
MPO homogenate buffer (see Recipes)
Note: All salts and other chemicals are from Sigma-Aldrich unless otherwise noted (however, any source for such chemicals is probably okay to use).
Equipment
1-0 braided silk material, bulk spool (e.g. Look catalog number: MBJF210)
12 mm x 75 mm x 1 mm microscope slides
2 x 2 gauze sponges, 8-ply (VWR International, catalog number: 82004-740 )
Sterile 4 x 4 gauze sponges, 8-ply (VWR International, catalog number: 82004-742 )
6-0 monofilament polypropylene suture with P-13 cutting needle (e.g. Syneture, catalog number: SP-5695 )
60° incline dissection board (homemade out of plexiglass)
Syringes (0.5, 1, 3, 5, 20 cc (cubic centimeter))
Needles (14, 20, 22, 26, 29 gauge)
Tracheal cannula (23 gauge x 1/2” stainless steel tubing adapter) (Becton, Dickinson and Company, catalog number: 408213 )
3” curved serrated forceps (2)
3” curved tissue (“toothed”) forceps
1.8 ml microfuge tubes
12 x 75 mm polystyrene tubes
22 x 22 mm #1.5 coverslips
4” curved micro dissecting scissors
37 °C water bath
Disposable skin stapler (e.g. 3M, model: DS-25 )
Hemocytometer or Coulter counter (e.g. Beckman Coulter, model: MultiSizer III )
Cytocentrifuge with cytospin funnels (e.g. Shandon CytoSpin®)
Tissue homogenizer (e.g. BrinkmannTMPolytronTM, model: PT2000 )
Probe sonicator (e.g. Branson Sonifier®, model: 450 )
Procedure
"Injury" procedure
Fill a 0.5 cc syringe with 22 gauge needle with 0.2 ml air “chaser” then “injury” solution (rat: 1 cc syringe with 14 gauge needle with 0.5 ml air, rabbit: 5 cc syringe with 14 gauge needle with 2 ml air).
Induce anesthesia in a chamber with 3-4% isoflurane in oxygen delivered at 1-2 L/min (rabbit: 30 mg/kg ketamine intramuscularly prior to 2% isoflurane) (Figures 1 and 2). Suspend mouse by its front teeth with a 1-0 suture strand on a 60° incline dissection board and maintain anesthesia with 2-2.5% isoflurane administration with nose cone.
Figure 1. Lab bench set-up for surgical procedures. Procedures are performed in a fume hood with charcoal filtering for anesthetic gas scavenging that contains: small animal anesthetic gas exposure chamber (front right), lamps, and the plexiglass 60° incline dissection board (middle of hood). On the left of the picture, on a floor stand outside the hood, is the anesthetic vaporizer for delivering the isoflurane vapor.
Figure 2. Instruments used for aspiration injury procedure. Plexiglass 60° incline dissection board is shown with nose cone setup to deliver isoflurane during the procedure. The green hose delivers the isoflurane in 100% oxygen to an inner nose cone, whereas the larger bore blue hose provides vacuum for scavenging the anesthetic through the outer nose cone. Also shown are (left to right): a 5 cc syringe with 26 gauge needle containing 1.5 ml normal saline for the subcutaneous fluid injection, a 0.5 cc syringe with 22 gauge needle containing the injury solution and 0.2 ml air “chaser”, 3 curved dissecting forceps (2 serrated and 1 “toothed”), curved dissecting scissors, a vial of antiseptic antimicrobial solution with cotton-tipped applicator, and a 6” strand of 1-0 braided silk suture material. Missing from picture: disposable skin stapler.
Shave ventral neck, paint with topical antiseptic prep solution and remove excess with gauze.
Infiltrate future incision site with 100 μl 0.5% bupivacaine (provides local post-operative analgesia).
Cut a 2 cm longitudinal incision in the skin with scissors (Figure 3).
Figure 3. Neck incision. After suspending the mouse by its front incisors with a suture such that its nose is within the inner isoflurane nose cone (step A2), prepping the surgical site (steps A3 & A4), a 2 cm longitudinal incision is made with scissors and tissue forceps (step A5).
Expose trachea by blunt dissection with 2 curved toothless forceps (Figures 4, 5 and 6).
Figure 4. Tracheal exposure by blunt dissection. The fascia membrane is teased away with curved serrated forceps and the salivary glands are pulled to the side in order to expose the trachea (faint white vertical between the forceps) that is surrounded by paratracheal musculature (step A6).
Figure 5. Tracheal exposure by blunt dissection, continued. Grasp one side of the paratracheal musculature and pull it to the side while rubbing along the muscle longitudinally. This will result in a separation of the muscle fiber bundles and allow the muscle to be reflected to the side (step A6). Notice the inner and outer nose cone configuration for delivering the anesthetic and scavenging. Mouse’s nose has been positioned lower than usual for clarity.
Figure 6. Tracheal exposure by blunt dissection, continued. Use the two curved serrated forceps to reflect the paratracheal musculature, fully exposing the trachea (step A6).
Work curved forceps under trachea (Figure 7). and use to pull a 6" strand of 1-0 silk suture material through (Figure 8).
Figure 7. Work curved serrated forceps under trachea (step A7)
Figure 8. Use forceps to place suture under trachea (step A7)
Discontinue isoflurane administration by removing nose cone. Before instilling injury vehicle by the steps below, allow mouse’s plane of anesthesia to rise until it just starts reacting to a forceps toe pinch then instigate instillation steps quickly. Especially for the acid-containing injury vehicles, if the plane of anesthesia is too deep the animal will not begin to breathe spontaneously after the injury vehicle is instilled.
Lift trachea up with suture to facilitate inserting injury syringe needle into the trachea (2-3 cartilage rings below the larynx) with bevel facing surgeon (advance until bevel of needle is just past the trachea insertion point) (Figure 9). It is important that the needle be as parallel with the trachea as possible, otherwise, the needle can easily pierce the other side of the trachea. This will result in the injury vehicle not being injected into the lungs and a high probability that the animal will not survive.
Figure 9. Use suture to facilitate injecting injury solution into trachea. Remove nose cone apparatus from incline board to provide unrestricted access to upper section of trachea. Lift trachea up with suture to facilitate inserting injury syringe needle into the trachea (2-3 cartilage rings below the larynx). Notice bevel of needle facing surgeon. Advance needle until bevel of needle is just past the trachea insertion point (step A9). It is important that the needle be as parallel with the trachea as possible, otherwise, the needle can easily pierce the other side of the trachea. This will result in the injury vehicle not being injected into the lungs and a high probability that the animal will not survive. While assistant squeezes rib cage (to expel most of vital capacity), quickly instill syringe contents. Release chest just before the injection begins. This maneuver, along with the air “chaser” assures deposition of the injury vehicle into the distal lung (step A10).
While assistant squeezes rib cage (to expel most of vital capacity), quickly instill syringe contents. Release chest just before the injection begins. This maneuver, along with the air “chaser” assures deposition of the injury vehicle into the distal lung.
Leave animal on incline board until breathing commences and close incision with 2 staples (for rats and rabbits: trachea needle wound needs to be repaired with one 6-0 suture) (Figure 10).
Figure 10. Close neck skin incision with surgical staples (step A11)
Inject 1 ml (rat: 10 ml, rabbit: 20 ml) sterile NS subcutaneously into the scruff of the neck for fluid resuscitation. There is virtually no fluid loss during the procedure, but the animal will not drink for a while after the procedure and can dehydrate (Figure 11).
Figure 11. Fluid resuscitation. Inject 1 ml sterile NS subcutaneously into the scruff of the neck using a syringe with 26 gauge needle for fluid resuscitation (step A12).
Put into heated chamber (37 °C) perfused with 100% O2 until ambulatory (Figure 12).
Figure 12. Recovery chamber. Place mouse in recovery chamber that is continually perfused with 100% O2 (supplied by green hose) and maintained at 37 °C with heat lamp and temperature controller (notice thermistor probe in left chamber) until the mouse is ambulatory (step A13).
Harvest procedure (Figure 13)
Figure 13. Instruments and materials used for harvest. From left to right: two serrated dissecting forceps, tissue (“toothed”) forceps, dissecting scissors, 23 gauge stainless steel blue-hubbed cannula, 1 cc blood collection syringe with 26 gauge needle, bronchoalveolar lavage apparatus (top syringe filled with 5 ml HBSS without Ca2+, Mg2+, left syringe is for fluid collection), two 2 x 2 gauze sponges (bottom left) for retracting abdominal organs, and 4” strand of 1-0 braided silk suture (bottom, middle). Missing from picture: 5 cc syringe with 26 gauge needle containing 5 ml HBSS with Ca2+, Mg2+ for flushing pulmonary vasculature.
Anesthetize mouse with isoflurane in 100% O2.
When mouse is unresponsive, place on a dissecting board in supine position and continue isoflurane administration with a nose cone.
Using forceps and scissors, make a longitudinal incision up the abdomen to the xyphoid.
Make a latitudinal incision across the belly (Figure 14) and expose vena cava and abdominal aorta using gauze sponges to reflect abdominal organs (Figure 15).
Figure 14. Abdominal incision for collecting blood. Using forceps and scissors, make a longitudinal incision up the abdomen to the xyphoid and a latitudinal incision across the belly (steps B3 & B4).
Figure 15. Collect blood. Expose vena cava and abdominal aorta using gauze sponges to reflect abdominal organs and collect blood using a 1 cc syringe with 26 gauge needle from either vessel (steps B4 & B5).
Harvest blood by (used to assess systemic inflammatory responses (e.g. serum or plasma cytokine levels)):
Collect blood from abdominal aorta using 27 gauge needle on a 1 cc syringe (if plasma is to be isolated syringe must contain appropriate anticoagulant) (Figure 15).
The vena cava can be used to collect blood, but generally, not as much blood can be collected as from the aorta (Figure 15).
Dispense blood into 1.8 ml μ-fuge tube. Until blood can be processed, keep on ice if plasma will be prepared, or keep at room temperature if serum will be prepared. Process blood, as described below.
Transect vena cava/abdominal aorta to euthanize by exsanquination.
Flush pulmonary vasculature by (performed to remove the blood from the pulmonary circulation so measurements of compounds in the processed lung tissue reflect only values from the pulmonary compartment that do not include a systemic contribution):
Grasp xyphoid process with forceps, puncture diaphragm with tips of scissors to deflate lungs and carefully cut away ventral aspect of diaphragm (Figure 16).
Figure 16. Cut-away diaphragm. Grasp xyphoid process with forceps, puncture diaphragm with tips of scissors to deflate lungs and carefully cut away ventral aspect of diaphragm (step B6a).
Continue the longitudinal incision up the sternum and through the neck.
Cut away sternum to expose lungs (be careful not to puncture lungs) (Figure 17).
Figure 17. Perform sternotomy. Continue the longitudinal incision up the sternum and through the neck. Cut away sternum to expose lungs (be careful not to puncture lungs) (steps B6b-c).
Grasp apex of heart with toothless forceps and inject 5 ml (rat: 20 ml) 1x HBSS with Ca2+, Mg2+ (at 37 °C) into right ventricle (it will be on the left side of the heart since the animal is on its back) with 5 cc (rat: 20 cc) syringe + 26 gauge needle (Figure 18). The rate of injection should be fast enough to keep the heart “inflated”, but not so fast as to force the fluid out through the injection site. The calcium in the buffer, and its being at 37 °C, aids the heart to keep beating to facilitate flushing the pulmonary vasculature.
Figure 18. Flush pulmonary vasculature. Grasp apex of heart with serrated forceps (tissue forceps have a tendency to tear the heart tissue) and inject 5 ml 1x HBSS with Ca2+, Mg2+ (at 37 °C) into right ventricle (it is on the left side of the heart in this picture since the animal is on its back) with 5 cc syringe + 26 gauge needle. The rate of injection should be fast enough to keep the heart “inflated”, but not so fast as to force the fluid out through the injection site (step B6d).
Perform bronchoalveolar lavage (BAL) by (performed to collect cells and compounds secreted into the airspaces (i.e. alveoli and bronchial lumen) that can be assessed to determine the degree of pulmonary inflammation (i.e. neutrophil influx, various inflammatory cytokine levels (i.e. tumor necrosis factor-alpha (TNFα), interleukin-1beta (IL-1β), IL-6, macrophage inflammatory protein-2 (MIP-2), monocytic chemotactic protein-1 (MCP-1)), and lung injury (i.e. total protein or albumin concentration):
Expose trachea and work tip of curved forceps under it to pull a 4" strand of 1-0 silk suture through.
Insert a 23 gauge x 1/2” (rat: 14 gauge, rabbit: 3 mm ID, 4.5 mm OD) stainless steel cannula (rabbit: polyethylene catheter) into the trachea and secure with suture (Figures 19 & 20).
Figure 19. Insert tracheal cannula. Expose trachea and work tip of curved forceps under it to pull a 4" strand of 1-0 braided silk through, as described in the injury procedure (steps A6, A7 & A9). Use a 20 gauge needle to make a hole in the upper trachea and insert a 23 gauge 1/2” stainless steel cannula into the hole.
Figure 20. Secure tracheal cannula. Secure cannula in the trachea with the suture (steps B7a-b). Be sure the suture is tight and 2-3 mm below the insertion site so a good seal is made. Tip of cannula should be at least 3 mm before the carina (bifurcation of the trachea). If the 1/2” cannula is inserted just below the larynx, as depicted in the figure, the tip of the cannula will be in the proper position.
Connect lavage apparatus (2, 5 cc syringes connected to a 4-way stopcock) to catheter and slowly, lavage lungs with 5 x 1 ml (rat: 10 ml, rabbit: 50 ml) 1x HBSS without Ca2+, Mg2+ (at 37 °C) (Figures 21 & 22). The lack of calcium in the buffer facilitates harvesting of the airway cells by diminishing adherence.
Figure 21. Perform bronchoalveolar lavage (BAL)-injection. Connect lavage apparatus to cannula and slowly inject 1 ml of 1x HBSS with Ca2+, Mg2+ to insulflate lungs (step B7c).
Figure 22. Perform BAL-collection. Switch stopcock valve and slowly collect the injected lavage fluid. Repeat the injection and collection process for a total of 5 lung washings (step B7c).
Dispense collected BAL fluid (BALF) into appropriately sized tube, record recovered volume (by weighing), and put on ice until BALF processing, described below.
Harvest lungs by (performed to assess the pulmonary inflammatory state distinct from the airspaces, i.e., parenchymal and interstitial tissue):
Excise lungs, heart, and thymus, en bloc, by the trachea (Figure 23).
Figure 23. Harvest lungs, heart, and thymus, en bloc. Be sure clavicle has been cut away. Grasp cannula, trachea, and suture with fingers and cut trachea by larynx. While pulling up and out, work scissors dorsally under lungs and spread to disengage tissue connecting lungs and heart to thoracic cavity. Some cutting of connective tissue will be needed, as well as the esophagus and vessels to successfully remove the lungs, heart, and thymus, en bloc (step B8a).
Remove lung lobes by cutting lobe’s main stem bronchus at hilum (Figures 24 & 25), place in 2 ml cryovial, and flash freeze in liquid N2.
Figure 24. Harvest lung lobes. Collect individual lobes by using scissors and forceps to cut main stem bronchi at the lobe’s hilum (step B8b).
Figure 25. Mouse lung lobes. Clockwise from left: left lung lobe, right lung-cranial lobe, right lung-middle lobe, right lung caudal lobe, right lung-post-caval lobe.
Store at -80 °C until appropriate processing can be performed, described below.
Blood processing
Plasma
Centrifuge at 2,000 x g for 4 min at 4 °C.
Remove plasma with a pipet and dispense into 1.8 ml microfuge tube.
Store at -80 °C.
Serum
Incubate at RT for 30 min then overnight at 4 °C to allow clot to contract.
Centrifuge at 5,000 x g for 15 min at 4 °C.
Remove serum with a pipet and dispense into 1.8 ml microfuge tube.
Store at -80 °C.
BALF processing (keep samples on ice)
Spin BALF at 1,500 x g for 5 min at 4 °C.
Without disturbing pellet, collect supernatant with pipet (leave 50-100 μl residual volume of uncollected supernatant to prevent sucking-up cells) and dispense into equal volume aliquots in 1.8 ml microfuge tubes (the number and volume of the aliquots will depend on the assays that will be performed and should be designed to limit the amount of freeze/thaw cycles). Store at -80 °C.
Resuspend pellet in the residual volume.
Add 1 ml ammonium chloride lysis buffer (37 °C) and incubate for 2 min to lyse red blood cells.
Layer entire volume on 4 ml ice cold 2% BSA in PBS in a 12 x 75 mm PS tube.
Spin at 150 x g for 15 min at 4 °C.
Aspirate supernatant (containing cell debris and lysed red blood cells), resuspend pellet in residual volume, and add 1 ml PBS. Removing the cell debris and lysed red blood cells by centrifuging the cells through 2% BSA makes counting the cells much easier and more accurate.
Count cells on hemocytometer or Coulter counter.
Prepare cytospin slide for microscopic viewing by:
Add 5 x 104 white blood cells to 2% BSA in PBS in a cytospin funnel (300 μl total volume) attached to a microscope slide and filter card and spin at 28 x g (500 rpm) for 5 min.
Remove slides and allow to air dry (IMPORTANT: stain immediately when dry).
Stain with Diff Quik by:
Dip slides 5 x 1 sec in fixative, Solution I, Solution II (Diff-Quik kit), rinse with H2OMQ & air dry.
Mount slide with small drop of "Cytoseal 60" on cell spot and putting a 22 x 22 mm #1.5 coverslip on it (be sure no bubbles) and allow to air dry.
Determine percentage of macrophages, neutrophils, lymphocytes, and eosinophils by light microscopy.
Lung processing (Lungs can be processed in a number of different ways depending on what cellular compartment (e.g. whole lung homogenate, nuclear fraction, cytosolic fraction, etc.) and molecular species (e.g. protein, mRNA, etc.) are to be assayed and what assay techniques will be utilized (e.g. ELISA, Western blot, PCR, etc.). This procedure produces a lung homogenate supernatant that can be assayed for various cytokine protein levels by ELISA, as well as an extraction of myeloperoxidase from the lung homogenate pellet that can be assayed for enzymatic activity as a surrogate marker of neutrophil infiltration)
Thaw frozen lungs and transfer to a round bottom centrifuge capable of withstanding 40,000 x g. Weigh tube before adding lungs then weigh the tube + lungs.
Add enough cytokine homogenate buffer to bring the weight of the lungs + buffer to 3 g (rat: 10 g).
Homogenize tissue with Polytron homogenizer with tube on ice to prevent heating of sample. Pick out any tissue caught in homogenizer blades with forceps. Rinse homogenizer with 70% EtOH and then sterile H2O between samples.
Centrifuge homogenate at 40,000 x g for 10 min at 4 °C.
Dispense supernatant into equal volume aliquots in 1.8 ml microfuge tubes and store at -80 °C.
Add 2 ml MPO buffer to pellet and resuspend by vortexing.
Store at -80 °C (in same tube) until MPO extraction can be performed.
MPO extraction procedure
Quick thaw sample in 37 °C water bath.
Sonicate for 1 min, on ice to prevent sample heating, on maximum output at 50% duty cycle.
Incubate at 55 °C for 2 h.
Centrifuge at 40,000 x g for 15 min at 4 °C.
Dispense into 0.5 ml aliquots in 1.8 ml microfuge tubes and store at -80 °C until MPO activity can be assessed
Recipes
100 mg/ml mouse (rat) gastric particles
Harvest stomach from freshly necropsied mouse (rat) first thing in the morning and put in 50 ml tube on ice. Harvesting early in the morning assures a full stomach.
In laminar flow hood
Put stomach in Petri dish, cut longitudinally with scissors.
Place stomach contents in a clean, 50 ml centrifuge tube.
Add 10 ml sterile normal saline (NS) and vortex vigorously for 15 sec.
Pour particle suspension through 8 layers of sterile gauze into a beaker.
Use an additional 10 ml sterile NS to transfer residual particles in tube to gauze.
Wring gauze to collect absorbed fluid.
Transfer filtrate to a clean, 50 ml centrifuge tube.
Spin filtrate at 5,000 x g for 5 min at 4 °C.
Discard supernatant, and resuspend in 25 ml, sterile NS.
Repeat steps 1c & 1d.
Transfer particle suspension to an Erlenmeyer flask and autoclave 121 °C, 121 psi for 30 min (use an additional 25 ml, sterile NS to transfer residual particles).
Transfer cooled, sterile particle suspension to a pre-weighed 50 ml centrifuge tube.
Spin filtrate at 5,000 x g for 5 min at 4 °C.
Decant supernatant and stand tube inverted on KIMWipe to remove all liquid.
Re-weigh tube+particle pellet to determine particle wet weight.
Add sterile NS to 100 mg/ml (particle wet weight/total volume).
Store at 4 °C for up to 3 weeks until used for injury.
Acid injury solution (acid)
Normal saline (NS), sterile
9.44 ml
1 N HCl, sterile
562 μl
Adjust pH = 1.25 with sterile 1 N HCl
Make fresh
Gastric particles injury solution (part.)
Normal saline (NS), sterile
9 ml
100 mg/ml gastric particles in NS
1 ml (10 mg/ml)
Make fresh.
Acid + particles injury solution (acid + part.)
Normal saline (NS), sterile
8.44 ml
1 N HCl, sterile
562 μl
100 mg/ml gastric particles in NS
1 ml (10 mg/ml)
Adjust pH = 1.25 with sterile 1 N HCl
Make fresh
Phosphate buffered saline (PBS), pH 7.2
NaCl (58.44)
8 g (137 mM)
Na2HPO4 (141.96)
1.15 g (8.1 mM)
KCl (74.56)
200 mg (2.7 mM)
KH2PO4 (136.09)
200 mg (1.5 mM)
H2OMQ to 1 L
Adjust pH = 7.2, filter sterilize
Stored at room temperature
Ammonium chloride lysis buffer
NH4Cl (53.49)
4.13 g (154 mM)
KHCO3 (100.1)
500 mg (10 mM)
EDTA, tetrasodium salt (380.2)
18.5 mg (0.1 mM)
Mix powders together, thoroughly
Dispense equal amounts into 10, 50 ml centrifuge tubes (0.46 g/tube)
On day of use, add 50 ml H2OMQ and mix until dissolved
Discard any unused solution
Lung homogenate buffer
NaCl (58.44)
8.77 g (150 mM)
Tris base (121.14)
1.82 g (15 mM)
CaCl2.2H2O (147.02)
147 mg (1 mM)
MgCl2.6H2O (203.3)
203 mg (1 mM)
H2OMQ to 1 L
Adjust pH = 7.4, autoclave at 121 °C, 15 psi, for 15 min
Stored at 4 °C
At time of use, add 1/100th volume of 100x protease inhibitor cocktail to volume of lung homogenate buffer needed for the day’s processing
50 mM potassium phosphate buffer, pH = 6.0
KH2PO4 (136.01)
3.4 g (50 mM)
H2OMQ to 500 ml
Adjust pH = 6.0 with 2 M NaOH
Stored at room temperature
MPO homogenate buffer
Hexadecyltrimethylammonium bromide (364.5)
2 g (0.5%)
EDTA (372.24)
744 mg (5 mM)
50 mM potassium phosphate buffer, pH = 6.0
400 ml
Stored at room temperature (Do not refrigerate)
Acknowledgments
The work presented here was supported by NIH grant HL048889, “Pathogenesis of Aspiration Pneumonitis” to Paul R Knight, M.D., Ph.D. and Bruce A. Davidson, Ph.D. To cite this protocol please also use the following reference: Davidson et al. (2013).
References
Mouse models
Davidson, B. A., Vethanayagam, R. R., Grimm, M. J., Mullan, B. A., Raghavendran, K., Blackwell, T. S., Freeman, M. L., Ayyasamy, V., Singh, K. K., Sporn, M. B., Itagaki, K., Hauser, C. J., Knight, P. R. and Segal, B. H. (2013). NADPH oxidase and Nrf2 regulate gastric aspiration-induced inflammation and acute lung injury. J Immunol 190(4): 1714-1724.
Guo, W. A., Davidson, B. A., Ottosen, J., Ohtake, P. J., Raghavendran, K., Mullan, B. A., Dayton, M. T. and Knight, P. R., 3rd (2012). Effect of high advanced glycation end-product diet on pulmonary inflammatory response and pulmonary function following gastric aspiration. Shock 38(6): 677-684.
Hutson, A. D., Davidson, B. A., Raghavendran, K., Chess, P. R., Tait, A. R., Holm, B. A., Notter, R. H. and Knight, P. R. (2006). Statistical prediction of the type of gastric aspiration lung injury based on early cytokine/chemokine profiles. Anesthesiology 104(1): 73-79.
Raghavendran, K., Davidson, B. A., Mullan, B. A., Hutson, A. D., Russo, T. A., Manderscheid, P. A., Woytash, J. A., Holm, B. A., Notter, R. H. and Knight, P. R. (2005). Acid and particulate-induced aspiration lung injury in mice: importance of MCP-1. Am J Physiol Lung Cell Mol Physiol 289(1): L134-143.
Segal, B. H., Davidson, B. A., Hutson, A. D., Russo, T. A., Holm, B. A., Mullan, B., Habitzruther, M., Holland, S. M. and Knight, P. R., 3rd (2007). Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice. Am J Physiol Lung Cell Mol Physiol 292(3): L760-768.
Rat models
Davidson, B. A., Knight, P. R., Helinski, J. D., Nader, N. D., Shanley, T. P. and Johnson, K. J. (1999). The role of tumor necrosis factor-alpha in the pathogenesis of aspiration pneumonitis in rats. Anesthesiology 91(2): 486-499.
Davidson, B. A., Knight, P. R., Wang, Z., Chess, P. R., Holm, B. A., Russo, T. A., Hutson, A. and Notter, R. H. (2005). Surfactant alterations in acute inflammatory lung injury from aspiration of acid and gastric particulates. Am J Physiol Lung Cell Mol Physiol 288(4): L699-708.
Kennedy, T. P., Johnson, K. J., Kunkel, R. G., Ward, P. A., Knight, P. R. and Finch, J. S. (1989). Acute acid aspiration lung injury in the rat: biphasic pathogenesis. Anesth Analg 69(1): 87-92.
Knight, P. R., Druskovich, G., Tait, A. R. and Johnson, K. J. (1992). The role of neutrophils, oxidants, and proteases in the pathogenesis of acid pulmonary injury. Anesthesiology 77(4): 772-778.
Knight, P. R., Rutter, T., Tait, A. R., Coleman, E. and Johnson, K. (1993). Pathogenesis of gastric particulate lung injury: a comparison and interaction with acidic pneumonitis. Anesth Analg 77(4): 754-760.
Knight, P. R., Davidson, B. A., Nader, N. D., Helinski, J. D., Marschke, C. J., Russo, T. A., Hutson, A. D., Notter, R. H. and Holm, B. A. (2004). Progressive, severe lung injury secondary to the interaction of insults in gastric aspiration. Exp Lung Res 30(7): 535-557.
Nader-Djalal, N., Knight, P. R., Davidson, B. A. and Johnson, K. (1997). Hyperoxia exacerbates microvascular lung injury following acid aspiration. Chest 112(6): 1607-1614.
Nader-Djalal, N., Knight, P. R., 3rd, Thusu, K., Davidson, B. A., Holm, B. A., Johnson, K. J. and Dandona, P. (1998). Reactive oxygen species contribute to oxygen-related lung injury after acid aspiration. Anesth Analg 87(1): 127-133.
Nader, N. D., Knight, P. R., Bobela, I., Davidson, B. A., Johnson, K. J. and Morin, F. (1999). High-dose nitric oxide inhalation increases lung injury after gastric aspiration. Anesthesiology 91(3): 741-749.
Nader, N. D., Knight, P. R., Davidson, B. A., Safaee, S. S. and Steinhorn, D. M. (2000). Systemic perfluorocarbons suppress the acute lung inflammation after gastric acid aspiration in rats. Anesth Analg 90(2): 356-361.
Nader, N. D., Davidson, B. A., Tait, A. R., Holm, B. A. and Knight, P. R. (2005). Serine antiproteinase administration preserves innate superoxide dismutase levels after acid aspiration and hyperoxia but does not decrease lung injury. Anesth Analg 101(1): 213-219, table of contents.
Rotta, A. T., Shiley, K. T., Davidson, B. A., Helinski, J. D., Russo, T. A. and Knight, P. R. (2004). Gastric acid and particulate aspiration injury inhibits pulmonary bacterial clearance. Crit Care Med 32(3): 747-754.
Raghavendran, K., Davidson, B. A., Knight, P. R., Wang, Z., Helinski, J., Chess, P. R. and Notter, R. H. (2008). Surfactant dysfunction in lung contusion with and without superimposed gastric aspiration in a rat model. Shock 30(5): 508-517.
Raghavendran, K., Davidson, B. A., Huebschmann, J. C., Helinski, J. D., Hutson, A. D., Dayton, M. T., Notter, R. H. and Knight, P. R. (2009). Superimposed gastric aspiration increases the severity of inflammation and permeability injury in a rat model of lung contusion. J Surg Res 155(2): 273-282.
Raghavendran, K., Davidson, B. A., Hutson, A. D., Helinski, J. D., Nodzo, S. R., Notter, R. H. and Knight, P. R. (2009). Predictive modeling and inflammatory biomarkers in rats with lung contusion and gastric aspiration. J Trauma 67(6): 1182-1190.
Shanley, T. P., Davidson, B. A., Nader, N. D., Bless, N., Vasi, N., Ward, P. A., Johnson, K. J. and Knight, P. R. (2000). Role of macrophage inflammatory protein-2 in aspiration-induced lung injury. Crit Care Med 28(7): 2437-2444.
Rabbit model
Knight, P. R., Kurek, C., Davidson, B. A., Nader, N. D., Patel, A., Sokolowski, J., Notter, R. H. and Holm, B. A. (2000). Acid aspiration increases sensitivity to increased ambient oxygen concentrations. Am J Physiol Lung Cell Mol Physiol 278(6): L1240-1247.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Davidson, B. A. and Alluri, R. (2013). Gastric Aspiration Models. Bio-protocol 3(22): e968. DOI: 10.21769/BioProtoc.968.
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Category
Immunology > Animal model > Mouse
Immunology > Animal model > Rabbit
Immunology > Animal model > Rat
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969 | https://bio-protocol.org/en/bpdetail?id=969&type=0 | # Bio-Protocol Content
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Immunocytochemical Detection of Recombinant Biomphalysin on Schistosoma mansoni Sporocysts
David Duval
RG Richard Galinier
JP Julien Portela
G Guillaume Mitta
BG Benjamin Gourbal
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.969 Views: 8523
Reviewed by: Fanglian He Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in PLOS Pathogens Mar 2013
Abstract
Schistosomiasis, or bilharzia, is a tropical disease caused by worms of the genus Schistosoma which infect about 200 million people. The life cycle of the parasite requires Biomphalaria, a specific genus of freshwater snails, as intermediate. Using an interactome approach employing B. glabrata plasma and S. mansoni primary sporocyst extracts, we identified a new cytolytic protein called Biomphalysin that displays similarities to members of the β-PFT superfamily known to form channels in targeted membranes. To investigate its mechanism of action, we produced a recombinant protein flanked by an N-terminal 6 histidine tag. Then, we investigated the ability of Biomphalysin to interact with the sporocyst tegument. This optimized protocol describes an immunocytochemical procedure to detect histidine tagged recombinant protein on the sporocyst tegumental membrane.
Keywords: Interaction host/parasite Immunity Biomphalaria Schistosoma Effector
Materials and Reagents
Paraformaldehyde (PAF) (Sigma-Aldrich, catalog number: 158127 )
PBS (Sigma-Aldrich, catalog number: P4417 )
BSA (Sigma-Aldrich, catalog number: A3803 )
poly-D-Lysine–coated slides (culture slides) (BD Biosciences, Falcon®, catalog number: 354632 )
Mouse anti-HisG monoclonal antibody (Life Technologies, catalog number: R940-25 )
Alexa Fluor 594 goat anti-mouse IgG (Life Technologies, catalog number: A 110005 )
Dako fluorescent mounting medium (Dako, catalog number: S3023 )
Primary sporocysts of S. mansoni (Brazilian strain) used for immunocytochemical experiments were obtained by transferring miracidia to Chernin’s balanced salt solution (CBSS) and maintaining at 26 °C under normoxic conditions for 24 h (Yoshino and Laursen, 1995). Then, 100 primary sporocysts were incubated for 1 h with 30 nM of recombinant Biomphalysin protein (Galinier et al., 2013). As negative control, 100 sporocysts were used without having been treated with recombinant biomphalysin.
N terminal His(6)-tagged biomphalysin was expressed in vitro using the Rapid Translation System (RTS 500 Wheat Germ CECF Kit) (5 PRIME, catalog number. 2402500 )
4% PAF (see Recipes)
PBS/3% BSA (see Recipes)
PBS/1% BSA (see Recipes)
Anti- HisG antibody 1:500 (see Recipes)
Anti-mouse IgG 1:1,000 (see Recipes)
Equipment
Coverslip 24 x 60 mm (VWR International, catalog number: 631-1575 )
BD BioCoatTM Poly-D-Lysine 8-well CultureSlides (Becton, Dickinson and Company, catalog number: 354632)
Eppendorf centrifuge (Eppendorf , model: 5810R )
Swinging agitator (Fisher scientific, model: 10758995 )
Fluorescence confocal laser-scanning microscope (ZEISS, model: LSM 700 )
Procedure
Collect 100 sporocysts and transfer to culture slide coated with poly-D-Lysine.
Centrifuge culture slide at 800 x g for 2 min.
Aspirate supernatant and fix sporocysts with 200 μl 4% PAF during 1 h at room temperature.
Centrifuge culture slide at 800 x g for 2 min and aspirate supernatant.
Wash twice with 200 μl PBS and repeat step 4.
Add 200 μl PBS/3% BSA and incubate for 2 h at room temperature without agitation.
Centrifuge culture slide at 800 x g for 2 min and aspirate supernatant.
Add 200 μl anti-His antibody diluted at 1:500 in PBS and incubate 1.5 h at room temperature with a shaking speed of approximately 12 oscillations per minute.
Wash three times with 200 μl PBS during 5 min with a shaking speed of 12 oscillations per minute and between wash, centrifuge culture slide.
Incubate sporocysts with 200 μl Alexa Flour 594-conjugated anti-mouse IgG diluted 1:1,000 in PBS/1% BSA for 45 min at room temperature and protect slides from light with a shaking speed of 12 oscillations per minute.
Repeat step 9.
Place 2 drops of Dako fluorescent mounting medium on slide and cover with a coverslip.
Leave mounted slide overnight at 4 °C and protect slides from light before observation.
Figure 1. Immunolocalization of recombinant Biomphalysin on S. mansoni sporocyst. Sporocysts were treated or not with recombinant Biomphalysin (A : positive and B ; negative control, respectively) and immunostained using anti-His primary IgG and Alexa Fluor 594-conjugated secondary antibodies. Images 1 and 3 were taken under Nomarski light microscopy; images 2 and 4 under using a fluorescence confocal laser-scanning microscope (Zeiss LSM 700).
Recipes
4% PAF
Dissolve 4 mg PAF in 100 ml PBS (heat to 60 °C for 1 h with stirring)
The cooled solution can be filtered and stored at -20 °C
PBS/3% BSA
0.3 mg BSA in 10 ml PBS
PBS/1% BSA
0.1 mg BSA in 10 ml PBS
Anti- His antibody 1:500
0.5 μl anti-his antibody in 250 μl PBS only
Anti-mouse IgG 1:1,000
0.5 μl anti-mouse IgG in 250 μl PBS/1%BSA
Acknowledgments
We thank Anne Rognon and Nathalie Arancibia for technical assistance. We thank all team members for their advice and fruitful discussions. This work was supported by funds from the Centre National de la Recherche (CNRS) and the Université de Perpignan Via Domitia (UPVD), and by a grant from the ANR (25390 Schistophepigen). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
Galinier, R., Portela, J., Mone, Y., Allienne, J. F., Henri, H., Delbecq, S., Mitta, G., Gourbal, B. and Duval, D. (2013). Biomphalysin, a new beta pore-forming toxin involved in Biomphalaria glabrata immune defense against Schistosoma mansoni. PLoS Pathog 9(3): e1003216.
Yoshino, T. P. and Laursen, J. R. (1995). Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells. J Parasitol 81(5): 714-722.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Immunology > Immune cell staining > Immunodetection
Biochemistry > Protein > Immunodetection
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This is an In Press version of the protocol that has not yet been assigned to an issue.
Peer-reviewed
E. coli Genomic DNA Extraction
Fanglian He
In Press
Published: Jul 20, 2011
DOI: 10.21769/BioProtoc.97 Views: 216810
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Abstract
This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits.
Materials and Reagents
Tris base (Calbiochem-Behring)
Proteinase K (Sigma-Aldrich)
Phenol\chloroform (1: 1) (EM Science)
200 proof ethanol (Pharmco-AAPER)
RNAase (Life Technologies, Invitrogen™)
Ethanol
SDS
EDTA
Tryptone
Yeast extract
NaCl
LB medium (see Recipes)
TE buffer (see Recipes)
Lysis buffer (see Recipes)
Equipment
Tabletop centrifuge (Eppendorf)
1.5 ml Eppendorf tube
Incubator
Gloves
Procedure
Transfer 1.5 ml of the overnight E. coli culture (grown in LB medium) to a 1.5 ml Eppendorf tube and centrifuge at max speed for 1min to pellet the cells.
Discard the supernatant.
Note: Remove as much of the supernatant as you can without disturbing the cell pellet.
Resuspend the cell pellet in 600 μl lysis buffer and vortex to completely resuspend cell pellet.
Incubate 1 h at 37 °C.
Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
Note: Do not vertex the tube—it can shear the DNA.
CAUTION: Phenol is a very strong acid that causes severe burns. Chloroform is a carcinogen. Wear gloves, goggles and lab coat, and keep tubes capped tightly. To be safe, work in the hood if possible.
Spin at max speed for 5 min at RT (all spins are performed at RT, unless indicated otherwise). There is a white layer (protein layer) in the aqueous: phenol/chloroform interface.
Carefully transfer the upper aqueous phase to a new tube by using 1 ml pipetman (to avoid sucking the interface, use 1 ml tip with wider mouth-cut 1 ml tip-mouth about ~2 mm shorter).
Steps 4-6 can be repeated until the white protein layer disappears.
To remove phenol, add an equal volume of chloroform to the aqueous layer. Again, mix well by inverting the tube.
Spin at max speed for 5 min.
Remove aqueous layer to new tube.
To precipitate the DNA, add 2.5 or 3 volume of cold 200 proof ethanol (store ethanol at -20 °C freezer) and mix gently (DNA precipitation can be visible).
Note: DNA precipitation may simply diffuse, which is normal. Keep the tube at -20 degree for at least 30 min (the longer the better) and then spin it down (see Steps 15-16). You should see DNA pellet. It looks transparency when it is wet and turns to white when it becomes dry.
Incubate the tube at -20 °C for 30 min or more.
Spin at max speed for 15 min at 4 °C.
Discard the supernatant and rinse the DNA pellet with 1 ml 70% ethanol (stored at RT).
Spin at max speed for 2 min. Carefully discard the supernatant and air-dry the DNA pellet (tilt the tube a little bit on paper towel). To be faster, dry the tube at 37 °C incubator.
Resuspend DNA in TE buffer.
Note: Large amounts of RNA will be present in the DNA sample. So, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to lysis buffer with a final concentration of 1 mg ml-1.
Check isolated Gemonic DNA on an agarose gel.
Note: we expect to see bands with smear patterns from high to low MW range, although most of DNA fragments are accumulated at high MW on the gel. So, if you see most of DNA fragments are small, very likely your DNA got degraded.
Recipes
LB medium
1% tryptone
0.5% yeast extract
200 mM NaCl
TE buffer
10 mM Tris-Cl (pH 8.0)
1 mM EDTA (pH 8.0)
Lysis buffer (10 ml)
9.34 ml TE buffer
600 ul of 10% SDS
60 μl of proteinase K (20 mg ml-1)
Acknowledgments
This protocol was adapted from Andrew Binns’ lab protocol collections at the University of Pennsylvania (USA).
References
Maniatis T., E.F. Fritsch, and J. Sambrook (1982). Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
Article Information
Copyright
© 2011 The Authors; exclusive licensee Bio-protocol LLC.
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Category
Microbiology > Microbial genetics > DNA
Molecular Biology > DNA > DNA extraction
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what it mean add equal volume?
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970 | https://bio-protocol.org/en/bpdetail?id=970&type=0 | # Bio-Protocol Content
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Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, a Bordetella pertussis Response Regulator, in vivo and in vitro
QC Qing Chen
AB Alice Boulanger
DH Deborah M. Hinton
SS Scott Stibitz
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.970 Views: 10828
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Microbiology Apr 2013
Abstract
Protein phosphorylation plays a central role in signal transduction in bacteria. However, separation and detection of the phosphorylated protein from its nonphosphorylated form remain challenging. Here we describe a method to detect phosphorylation of the Bordetella pertussis response regulator BvgA, which is phosphorylated at an aspartate residue (Boulanger et al., 2013). This method is based on the proprietary adduct, Phos-tagTM, a dinuclear metal complex, which together with Zn2+ or Mn2+, forms a complex with a phosphomonoesterdianion, such as the phosphorylated aspartate of a response regulator (Barbieri and Stock, 2008; Kinoshita and Kinoshita-Kikuta, 2011). For in vivo detection, B. pertussis cells are lysed in mild formic acid at 4 °C to minimize the disruption of the phospho-aspartate bond, and the phosphorylated BvgA is separated from its nonphosphorylated form by electrophoresis (SDS-PAGE) containing Phos-tagTM. Both forms of BvgA are subsequently detected by Western Blot analysis. Quantification of the level of phosphorylated BvgA formed after treatment with acetyl phosphate in vitro is also easily accomplished. Thus, this technique allows one to readily assess the levels of BvgA phosphorylation in B. pertussis and in E. coli under different laboratory conditions in vivo or after phosphorylation under varying reaction conditions in vitro (this research was supported in part by the Intramural Research Program of the NIH, NIDDK).
Keywords: Phos-tag Phosphorylation detection Response regulator
Materials and Reagents
Bordetella pertussis strain BP536 is the tet-resistant derivative of the clinical isolate Tohama I strain (laboratory inventory)
Formic acid (> 95%) (Sigma-Aldrich, catalog number: F0507 )
NaOH (Thermo Fisher Scientific, catalog number: SS255-1 )
Tris base (MP Biomedicals, catalog number: 819620 )
Bromophenol blue (Sigma-Aldrich, catalog number: B8026 )
Glycerol (Invitrogen, catalog number: 15514-011 )
Butanol (Sigma-Aldrich, catalog number: 15467-9 )
30% 29:1 Acrylamide/bis-acrylamide (Sigma-Aldrich, catalog number: A3574 )
10% SDS (Hoefer, catalog number: GR155-1 )
Phos-tagTM acrylamide (Wako Pure Chemical Industries, catalog number: AAL-107 )
Zn(NO3)2 (Sigma-Aldrich, catalog number: 228737 )
Ammonium persulfate (Sigma-Aldrich, catalog number: 3678 )
N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
0.5 M EDTA, pH 8.0 (Research Genetics, catalog number: 750009 )
MOPS (Fluka, catalog number: 69947 )
Sodium metabisulfite (Na2S2O5) (Sigma-Aldrich, catalog number: S9000 )
Glycine (Sigma-Aldrich, catalog number: G7126-1KG )
Methanol (EMD Millipore, catalog number: MX0485-7 )
PBS buffer (Gibco, catalog number: 2014-10 )
BactoTM proteose peptone (Becton, Dickinson and Company, catalog number: 211684 )
DifcoTM Bordet Gengou agar base (Becton, Dickinson and Company, catalog number: 248200 )
Defibrinated sheep blood (LAMPIRE® Biological Labs, catalog number: 7239001 )
Lithium potassium acetyl phosphate (Sigma-Aldrich, catalog number: A0262-500MG )
Colloidal Blue staining kit (Life Technologies, catalog number: LC6025 )
Tween-20 (Bio-Rad Laboratories, catalog number: M3524 )
Non-fat dry milk (Giant Food Store, catalog number: 688267078330 )
Monoclonal anti-BvgA antibody (laboratory inventory)
HRP-conjugated Secondary Antibody (Santa Cruz, catalog number: SC2005 )
Amersham ECL Primer Western Blotting Detection System (General Electric Company, catalog number: RPN2232 )
PVDF filter (Invitrogen, catalog number: LC6025 )
BSA (Albumin from bovine serum) (Sigma-Aldrich, catalog number: A2153 )
1 M formic acid (see Recipes)
5 N NaOH (see Recipes)
1 M Tris-Cl solutions (see Recipes)
1% Bromophenol blue (see Recipes)
5x Loading Solution (see Recipes)
Water-saturated butanol (see Recipes)
5 mM Phos-tagTM acrylamide (see Recipes)
10 mM Zn(NO3)2 (see Recipes)
10% APS (see Recipes)
1x MOPS Running Buffer, pH 7.8 (see Recipes)
Transfer Buffer (see Recipes)
BG agar plates (see Recipes)
200 mM acetyl phosphate (see Recipes)
1% or 5% BSA in PBS (see Recipes)
0.05% Tween-20 in PBS (see Recipes)
1% Non-fat milk in PBS (see Recipes)
Equipment
Polyester-tipped applicator (Puritan Medical, catalog number: 25-806 1PD )
Spectrophotometer
Mini gel cassettes (1.0 mm) (Invitrogen, catalog number: NC2010 )
10, 12 or 15-well combs (1.0 mm) (Invitrogen, catalog number: NC3015 )
XCell SureLock® Mini-Cell (Invitrogen, catalog number: EI0001 )
Platform shaker
Eppendorf microfuge
Bio-Rad Mini-Protean II (Bio-Rad Laboratories)
Procedure
Whole cell lysate preparation for in vivo detection of BvgA phosphorylation
Note: This protocol has been successfully used with another gram-negative bacteria, E. coli.
To collect bacteria sample, Bordetella pertussis strain BP536, grown at 37 °C for 2 days on BG agar plate, is swabbed from the plate with a polyester-tipped applicator and resuspended in 1.5 ml of PBS.
An aliquot from step A1 is used to determine the OD600 reading.
A 0.3 ml aliquot from step A1 is centrifuged at 15,600 x g in Eppendorf microfuge for 1 min at room temperature. The supernatant is removed, and the resulting pellet is frozen in dry ice. It can be used directly or stored at -80 °C.
The frozen pellet is treated as follows.
Note: Volumes are based on an OD600 reading of 0.5 and a pellet obtained from 0.3 ml, one should adjust the volumes proportionally based on the determined OD600.
First, 33 μl of ice-cold 1 M formic acid is added, and the pellet is disrupted by pipetting repeatedly. Immediately, a freshly made ice-cold solution (27 μl) containing 2 μl of 5 N NaOH (to neutralize the acid), 10 μl H2O, and 15 μl of 5x Loading Solution is added.
Note: The color of the solution turns yellow due to the acid, but the bromophenol blue changes back to blue once it enters the gel.
4 μl of the resulting cell lysate are loaded onto the Phos-tagTM gel for electrophoresis.
Note: Do not boil the treated cell lysate; the lysate should be kept on ice before loading.
Phos-tagTM SDS-PAGE gel electrophoresis (Recipe makes one mini gel.)
4% stacking gel
30% Acrylamide/bis-acrylamide
417.5 μl
1 M Tris pH 6.8
1,093 μl
10% SDS
31.3 μl
10% APS (freshly prepared)
25 μl
TEMED
5 μl
H2O
1,570 μl
10% resolving gel
30% Acrylamide/bis-acrylamide
2,085 μl
1 M Tris pH 6.8
2,185 μl
10% SDS
62.5 μl
5 mM Phos-tagTM
93.8 μl
10 mM Zn(NO3)2
93.8 μl
10% APS (freshly prepared)
25 μl
TEMED
5 μl
H2O
1,715 μl
For resolving gel: Acrylamide, Tris, SDS, Phos-tagTM, Zn(NO3)2, and H2O are mixed. 10% APS and TEMED are added, and the solution is immediately poured into the gel cassette. The solution is then overlaid with 1 ml of water-saturated butanol. The resolving gel polymerizes in ~40 min at room temperature.
The butanol is poured off of the polymerized resolving gel, and the gel surface is then washed with water. Excess water is removed using absorbent paper.
For stacking gel: Acrylamide, Tris, SDS, and H2O are mixed. 10% APS and TEMED are added, and the solution is immediately poured into the gel cassette. The comb is quickly inserted (avoiding the introduction of air bubbles) and the stacking gel is allowed to polymerize for 20 min at room temperature.
After insertion of the gel cassette into the electrophoresis apparatus, the ice-cold 1x MOPS Running Buffer is added. The entire apparatus is then placed into an ice-filled bucket and the bucket is placed on a platform shaker so that it can be gently rotated until the temperature of the Running Buffer in the chamber has cooled to 4 °C. This typically takes at least 30 min.
Note: Place only one gel cassette in one XCell SureLock® Mini-Cell to ensure that the temperature remains cold during electrophoresis.
The shaker is turned off and the samples are loaded using a pipetman.
After resumption of gentle shaking, electrophoresis is performed with constant 100 V for 30 min, 120 V for 30 min, and then 150 V until the bromophenol blue dye is ~ 1 cm from the bottom of the gel. (Temperature within the inner gel chamber will rise to 10 °C to 15 °C.)
For Western blot analysis, the gel is washed with 100 ml Transfer Buffer containing 1 mM EDTA for 10 min to remove Zn2+ from the gel, followed by washing again with 100 ml Transfer Buffer for 20 min.
The gel is transferred to a PVDF filter, which was previously rinsed with methanol and then with Transfer Buffer, in a Bio-Rad Mini-Protean II apparatus filled with ice-cold Transfer Buffer at 100 constant voltage for 1 h at 4 °C.
The antibody detection of BvgA is carried out at room temperature as follows: After the transfer, the PVDF filter is blocked by washing with 5% BSA in PBS for 1 h, and then incubated with monoclonal anti-BvgA antibody (1:5,000) in 1% BSA in PBS for 1 h. Three 10-min washes with 0.05% Tween-20 in PBS are conducted before incubation with HRP-conjugated Secondary Antibody (1:2,000) in 1% non-fat milk in PBS for 1 h. The PVDF filter is washed three times for 10 min, each, with 0.05% Tween-20 in PBS, and then developed with Amersham ECL Primer Western Blotting Detection System, according to the manufacture’s instruction.
Separation of in vitro phosphorylated purified protein
To phosphorylate in vitro, 9 μl purified BvgA (from ~1 to 25 pmol) is mixed with 1 μl 200 mM acetyl phosphate on ice and then incubated for the desired amount of time at 37 °C.
Reaction is stopped by placing the sample on dry ice.
Sample is mixed with 2.5 μl 5x Loading Solution (final concentration of 1x), separated on PhostagTM acrylamide gel, washed, and detected by Western analysis as described above.
Alternately, gel can be stained. In this case, the gel is first washed 3 times in 100 ml of water for 10 min. The gel is then treated with Colloidal Blue following manufacturer’s instructions.
Note: For best results, use either Colloidal Blue or classic Coomassie blue staining rather than other types of protein stains.
Note: The optimal level of purified protein for loading is ~ 1 pmol for Western analysis and ~25 pmol for gel staining
Figure 1. Separation of phosphorylated BvgA from unphosphorylated BvgA by Phos-tagTM SDS-PAGE, analyzed by Western blot. First lane shows the in vivo lysate of B. pertussis, which contains both phosphorylated BvgA (BvgA~P) and nonphosphorylated BvgA (BvgA). Lanes 2 and 3 show purified BvgA, which has been phosphorylated in vitro by the addition of acetyl phosphate or incubated in the absence of acetyl phosphate, respectively.
Recipes
1 M formic acid
5 ml of formic acid (> 95%) in 95 ml of H2O
Stored at 4 °C
5 N NaOH
5 ml 10 N NaOH
5 ml H2O
Stored at 4 °C
1 M Tris-Cl solutions (for 1 L)
121.14 g Tris base adjusted to pH 6.8 or to pH 8.0 with HCl at 4 °C
Filter sterilized
1% Bromophenol blue
1 g bromophenol blue in 100 ml H2O
5x Loading Solution
1% SDS
65 mM Tris-Cl (pH 6.8)
25% glycerol
0.02% bromophenol blue
Stored at 4 °C
Water-saturated butanol
4 ml water added to 40 ml butanol
Mix by shaking
Butanol will be the top layer
5 mM Phos-tagTM acrylamide
10 mg Phos-tagTM acrylamide dissolved in 100 μl methanol
Solution is pipetted repeatedly to dissolve the acrylamide
Add 3.2 ml H2O
Solution is centrifuged at 2,000 x g for 10 min to remove white precipitate
Stored at 4 °C in dark
10 mM Zn(NO3)2
2.97 g Zn(NO3)2 dissolved in 1 L H2O
Filter sterilized
10% APS
1 g ammonium persulfate dissolved in 10 ml H2O
Solution is freshly prepared
1x MOPS Running Buffer, pH 7.8
100 mM Tris-CL, pH 7.8
100 mM MOPS, pH 7.8
0.1% SDS
5 mM sodium metabisulfite
For 1 L
12.1 g Tris base
20.9 g MOPS
10 ml 10% SDS
0.95 g sodium metabisulfite (Na2S2O5)
Solution is adjusted to pH 7.8 with 10 N HCl at 4 °C
Stored at 4 °C
Transfer Buffer
3 g Tris base
14.4 g glycine
200 ml of methanol
800 ml of H2O
BG agar plates
10 g DifcoTM Bordet Gengou agar base
3.3 g BactoTM proteose peptone
3.3 ml glycerol added to 350 ml H2O
The mixture is autoclaved
Cooled to 46 °C before adding 50 ml defibrinated sheep blood
Pour warm solution into petri dishes and allow to cool to room temperature.
Stored at 4 °C
200 mM acetyl phosphate
0.0039 g lithium potassium acetyl phosphate dissolved in 105.7 μl 20 mM Tris-Cl, pH 8 Solution is freshly made
Stored on ice
1% or 5% BSA in PBS
1 g or 5 g BSA
100 ml PBS
0.05% Tween-20 in PBS
0.05 ml of Tween-20
100 ml of PBS
1% non-fat milk in PBS
1 g non-fat dry milk
100 ml of PBS
Acknowledgments
This protocol is based on the previously published paper Boulanger et al. (2013). The research was supported in part by the Intramural Research Program of the NIH, NIDDK.
References
Barbieri, C. M. and Stock, A. M. (2008). Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents. Anal Biochem 376(1): 73-82.
Boulanger, A., Chen, Q., Hinton, D. M. and Stibitz, S. (2013). In vivo phosphorylation dynamics of the Bordetella pertussis virulence-controlling response regulator BvgA. Mol Microbiol 88(1): 156-172.
Kinoshita, E. and Kinoshita-Kikuta, E. (2011). Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling. Proteomics 11(2): 319-323.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Chen, Q., Boulanger, A., Hinton, D. M. and Stibitz, S. (2013). Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, a Bordetella pertussis Response Regulator, in vivo and in vitro. Bio-protocol 3(22): e970. DOI: 10.21769/BioProtoc.970.
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Category
Microbiology > Microbial signaling > Phosphorylation
Cell Biology > Cell signaling > Phosphorylation
Biochemistry > Protein > Modification
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971 | https://bio-protocol.org/en/bpdetail?id=971&type=0 | # Bio-Protocol Content
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Peer-reviewed
Transport Assays in Aspergillus nidulans
Emilia Krypotou
George Diallinas
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.971 Views: 8199
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Microbiology Apr 2013
Abstract
Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. These assays make use of A. nidulans germinating conidiospores, thus avoiding technical difficulties associated with the use of mycelia. The ease of construction genetic null mutants in this model fungus permits the rigorous characterization of any transporter in the absence of similar transporters with overlapping specificities, a common problem in relevant studies.
Keywords: Ascomycetes Fungi Kinetics Specifcity Transporter
Materials and Reagents
p-aminobenzoic acid (Sigma-Aldrich, catalog number: P5669 )
d-Biotin (Sigma-Aldrich, catalog number: B4501 )
Calcium-D-pantothenate (Sigma-Aldrich, catalog number: 21210 )
Riboflavine (Sigma-Aldrich, catalog number: R4500 )
Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: P9755 )
KCl
MgSO4.7H2O
KH2PO4
Na2B4O7.10H2O
CuSO4.5H2O
FeO4P.4H2O
MnSO4.H2O
Na2MoO4.2H2O
ZnSO4.7H2O
NaOH
Tween 80 (Sigma-Aldrich, catalog number: P1754 )
Radiolabelled substrate
e.g. [8-3H]-xanthine, 22.8 Ci/mmol (Moravek Biochemicals, catalog number: MT537 )
[2,8-3H]-hypoxanthine, 27.7 Ci/mmol (Moravek Biochemicals, catalog number: MT700 )
[5-3H]-uracil, 23 Ci/mmol (Moravek Biochemicals, catalog number: MT610 )
Non radiolabelled substrate
e.g. Xanthine (Sigma-Aldrich, catalog number: X7375 )
Hypoxanthine (Sigma-Aldrich, catalog number: H9377 )
Uracil (Sigma-Aldrich, catalog number: U0750 )
Toluol (AppliChem GmbH, catalog number: A3393 )
Triton X-100
2,5-Diphenyloxazole (PPO) (Sigma-Aldrich, catalog number: D4630 )
1,4-bis (5-phenyloxazol-2-yl) benzene (POPOP) (Sigma-Aldrich, catalog number: P3754 )
Complete Media (CM) (see Recipes)
Minimal Media (MM) (see Recipes)
Scintillation Fluid (see Recipes)
Equipment
Petri dishes
Neubauer counting-chamber slide
Spatula
Orbital shaking incubator
Incubator at 37 °C
Nylon net filter 60 μm (Merck KGaA, catalog number: NY60 )
50 ml Falcon tubes
1.5 ml centifuge tubes
Centrifuge
Vortex
Scintillation vials
Scintillation counter
Heat block
Magnetic stirrer
Magnetic strirr bar
pH meter
Pasteur pipette
Software
GraphPad Prism software (Amillis et al., 2004)
Procedure
Inoculate a petri dish of CM with the strain of interest and let it reach full growth at 37 °C for 96 h.
Using a spatula transfer one quarter of the fully grown colony (~4 cm) in a 50 ml Falcon tube containing 2 ml of 0.01% v/v Tween 80 in water. This amount usually corresponds to 108 conidiospores. The accurate amount of conidiospores can be estimated using a Neubauer counting-chamber slide or by measuring viable conidiospores after standard serial dilutions and plating on CM.
Vortex well the sample for separating the conidiospores.
Inoculate a 100 ml flask containing 25 ml MM supplemented with appropriate carbon (C) (e.g. Glucose 1% w/v) and nitrogen (N) (e.g. NaNO3 10 mM) sources and necessary vitamins (e.g. D-biotin 0.02 μg/ml) with the conidiospores filtered through a Nylon net filter 60 μΜ. (All necessary supplements and concentrations for A. nidulans strains can be found at www.fgsc.net.)
Incubate for 3-5 h at 37 °C, shaking with 140 rpm, for the germinating conidiospores to reach a stage just before germ tube emergence. The time and temperature of incubation can change depending on the expression profile of the transporter of interest and the auxotrophic requirements of the strain. All transporters studied up to date in our lab (for example the purine/pyrimidine transporters UapA, UapC, AzgA, FcyB, FurD) reach maximum expression before germ tube emergence, driven by an unknown developmental control, irrespectively of the presence or absence of their substrates or other physiological conditions (Amillis et al., 2004; Vlanti and Diallinas, 2008; Amillis et al., 2007). In mycelia, transporter expression is very much dependent on physiological conditions (e.g. induction by substrates or/and N or C catabolite repression).
While conidiospores germinate, prepare the stock solution of radiolabeled (usually 3H or 14C) substrate of interest in water or MM so that for each assay 25 μl of the stock will be used.
Collect the conidiospores by centrifuging the culture in a 50 ml Falcon tube for 5 min, at 3,000 x g, room temperature.
Discard the supernatant and resuspend the pellet in 5 ml standard MM.
Distribute the spores in 75 μl aliquots in eppendorf tubes and use as many as needed. Conidiospore suspensions can be kept at 4 °C for at least 24 h without loss of transport activity.
Incubate conidiospore aliquots at 37 °C in a heat block for 5 min before addition of radiolabeled substrate.
Radiolabeled substrate is added for different periods of time. Most transporters show linearly increased activities for at least 1 min. For measuring initial uptake rates, which are necessary for determining Km and apparent Vmax values, the proper time of incubation must be defined for each transporter through a time-course experiment. For steady state substrate accumulation a period of 5 min is used. Usual time points are 10, 20, 30, 60 and 120 sec. For each time point, measurements are performed in triplicate. The temperature used for the incubation with the radiolabeled substrate depends on the transporter being studied at each experiment and the experiment being held, temperature for most experiments is 37 °C. The transport reaction is stopped by adding an equal volume (100 μl) of cold unlabeled substrate at 100-1,000 fold excess concentration, related to radiolabelled substrate, and direct transfer of the assay/eppendorf tube in an ice bucket.
Centrifuge the samples at 11,000 x g for 3 min at 4 °C.
Remove the supernatant through aspiration under vacuum using a Pasteur pipette. It is important to remove all the supernatant without losing any cells.
Wash the pellet of cells once with 1 ml ice cold MM and centrifuge at 11,000 x g for 3 min. Remove the supernatant as before.
Resuspend the pellet in 1 ml of scintillation fluid and put the eppendorf tubes into scintillation vials. Use a scintillation counter to measure substrate accumulation in the cells.
Analysis of transport measurements is performed using GraphPad Prism software. Radioactive counts should be converted to substrate concentration/min/conidiospores, based on the concentration and specific activity of the stock of radioactive substrate used.
For Km determination of a transporter, different substrate concentrations should be used, for a fixed incubation time, previously determined to reflect initial uptake rates. This is usually 1 min. The range of concentrations used is determined at first empirically. In the final experiment, at least three concentration points below and above the apparent Km value should be used. For each concentration point measurements are performed in triplicate.
The stock solutions are prepared using a mixture of fixed radiolabeled substrate and increasing concentrations of non-radiolabeled substrate, so that for each assay 25 μl of the stock will be used.
Terminate transport assays and perform measurements as described above.
For Ki determination of a transporter, the method is identical to the one for Km determination, but stock solutions are prepared using a mixture of fixed radiolabeled substrate and increasing concentrations of non-radiolabeled putative inhibitors. For each concentration point measurements are performed in triplicate.
Km and Vmax determination is carried out using typical Lineweaver-Burk plot analysis that is based on the Michaelis-Menten equation for enzyme kinetics V = Vmax[S]/(Km + [S]), where V is the reaction velocity (the reaction rate), Km is the Michaelis–Menten constant, Vmax is the maximum reaction velocity, and [S] is the substrate concentration. The Lineweaver-Burk plot depicts the linear expression of the previous equation which is transformed to the following: 1/V = (Km/Vmax).(1/[S]) + 1/Vmax. The data obtained by this experiment correspond to the apparent velocity of the transporter for each substrate concentration. Ki measurements are determined by estimating IC50 values (inhibitor concentration for obtaining 50% inhibition) of given substrate/inhibitor, using the formula Ki = IC50/1 + [S]/Km, where [S] is the fixed concentration of radiolabeled substrate used. Another way to analyse the data is by using the GraphPad Prism Software through a non linear regression curve fit and sigmoidal dose response analysis. The IC50 value corresponds to the Km/i of the transporter. Quality factors for the analysis result are: R2 which should be > 0.99 and the Hill co-efficient which should be approximately -1 for a transporter with one binding site.
The method described can be modified and adapted for most filamentous fungi that produce asexual spores, e.g. A. fumigatus. (Goudela et al., 2008)
Recipes
Complete Media (1 L)
Vitamin solution from 100x stock solution* 10 ml
Salt solution from 50x stock solution** 20 ml
Glucose 10 g
Peptone 2 g
Yeast Extract 1 g
Cas-amino- acids 1 g
Agar 10 g
Add water to 1 L final volume
Adjust the pH to 6.8 using NaOH
Autoclave for 20 min
*Vitamin stock solution
p-aminobenzoic acid 20 mg
d-biotin 1 mg
Calcium-D-pantothenate 50 mg
Riboflavin 50 mg
Pyridoxine 50 mg
Add water to 1 L final volume
**Salt stock solution
KCl 26 g
MgSO4.7H2O 26 g
KH2PO4 76 g
Trace elements 20x stock solution*** 50 ml
Add water to 1 L final volume
***Trace elements stock solution
Na2B4O7.10H2O 40 mg
CuSO4.5H2O 400 mg
FeO4P.4H2O 714 mg
MnSO4.H2O 728 mg
Na2MoO4.2H2O 800 mg
ZnSO4.7H2O 8 mg
Add water to 1 L final volume
Minimal Media
Salt solution from 50x stock solution* 20 ml
Add water until 1 L final volume
Adjust the pH to 6.8 using NaOH
Autoclave for 20 min
Scintillation Fluid (1 L)
Toluol 666 ml
PPO 2.66 g
POPOP 0.0066 g
2 h stirring in RT
Add Triton-X 100 333 ml
Overnight stirring
Acknowledgments
This protocol was adapted from the following publications: Diallinas et al. (1995); Koukaki et al. (2005); Meintanis et al. (2000); Tazebay et al. (1997). E.K. works in the laboratory of G.D, and is co-financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Thales, Investing in knowledge society through the European Social Fund.
References
Amillis, S., Cecchetto, G., Sophianopoulou, V., Koukaki, M., Scazzocchio, C. and Diallinas, G. (2004). Transcription of purine transporter genes is activated during the isotropic growth phase of Aspergillus nidulans conidia. Mol Microbiol 52(1): 205-216.
Amillis, S., Hamari, Z., Roumelioti, K., Scazzocchio, C. and Diallinas, G. (2007). Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter in Aspergillus nidulans. Mol Membr Biol 24(3): 206-214.
Diallinas, G., Gorfinkiel, L., Arst, H. N., Jr., Cecchetto, G. and Scazzocchio, C. (1995). Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes. J Biol Chem 270(15): 8610-8622.
Goudela, S., Reichard, U., Amillis, S. and Diallinas, G. (2008). Characterization and kinetics of the major purine transporters in Aspergillus fumigatus. Fungal Genet Biol 45(4): 459-472.
GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA. www.graphpad.com.
Koukaki, M., Vlanti, A., Goudela, S., Pantazopoulou, A., Gioule, H., Tournaviti, S. and Diallinas, G. (2005). The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway. J Mol Biol 350(3): 499-513.
Meintanis, C., Karagouni, A. D. and Diallinas, G. (2000). Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family. Mol Membr Biol 17(1): 47-57.
Tazebay, U. H., Sophianopoulou, V., Scazzocchio, C. and Diallinas, G. (1997). The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation. Mol Microbiol 24(1): 105-117.
Vlanti, A. and Diallinas, G. (2008). The Aspergillus nidulans FcyB cytosine-purine scavenger is highly expressed during germination and in reproductive compartments and is downregulated by endocytosis. Mol Microbiol 68(4): 959-977.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Krypotou, E. and Diallinas, G. (2013). Transport Assays in Aspergillus nidulans. Bio-protocol 3(22): e971. DOI: 10.21769/BioProtoc.971.
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Category
Microbiology > Microbial metabolism > Nutrient transport
Cell Biology > Cell-based analysis > Transport
Cell Biology > Cell-based analysis > Ion analysis
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972 | https://bio-protocol.org/en/bpdetail?id=972&type=0 | # Bio-Protocol Content
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Cotton Ovules Culture and Analysis
Jiafu Tan
FD Fenglin Deng
WT Wenxin Tang
JH Jie Han
GK Guo Kai
LT Lili Tu
Xianlong Zhang
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.972 Views: 10137
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Plant Physiology May 2013
Abstract
The cotton ovules culture was innovated by Beasley and Ting (1973), and named after them. It is a convenient system to analyze the effect of chemical or environmental treatment on fiber development directly on ovules. This protocol was generated according to previous published papers and our practical experience.
Keywords: Cotton Ovule culture Plant hormone Fiber
Materials and Reagents
Flowers (-1 to 1 DPA of flowers are easy to manipulate)
0.1% (w/v) HgCl2 (caution: HgCl2 is very dangerous. Please select an advantageous reagent to sterilize according to your lab condition. Otherwise 75% ethanol and N,N'-Dicyclohexylcarbodiimide (DCCS) could be the alternatives)
Toluidine blue O (Sigma-Aldrich, catalog number: T3260 )
Glacial acetic acid-ethanol-water (10:95:5, v/v)
Vitamins:
VB1(Vitamin B1, Thiamine) (Sigma-Aldrich, catalog number: T3902 )
VB6 (Vitamin B6, Pyridoxine) (Sigma-Aldrich, catalog number: P8666 )
VB3 (Vitamin B3, Nicotinic acid) (Sigma-Aldrich, catalog number: N0765 )
Inositol (Sigma-Aldrich, catalog number: I3011 )
IAA (Sigma-Aldrich, catalog number: I2886 )
GA3 (Sigma-Aldrich, catalog number: G7645 )
KH2PO4 (Sigma-Aldrich)
CaCl2.2H2O (Sigma-Aldrich)
MgSO4.7H2O (Sigma-Aldrich)
KNO3 (Sigma-Aldrich)
H3BO3 (Sigma-Aldrich)
Na2MoO4.2H2O (Sigma-Aldrich)
KI (Sigma-Aldrich)
CoCl2.6H2O (Sigma-Aldrich)
MnSO4.H2O (Sigma-Aldrich)
ZnSO4.7H2O (Sigma-Aldrich)
CuSO4.5H2O (Sigma-Aldrich)
FeSO4.7H2O (Sigma-Aldrich)
Na2EDTA (Sigma-Aldrich)
BT medium preparation (see Recipes)
Hormone preparation (see Recipes)
Working BT medium preparation (see Recipes)
Equipment
Erlenmeyer flask (50 ml)
1.5 ml microcentrifuge tubes
Spectrophotometer (Beckman Coulter, model: DU 800 ) or microplate reader (Tecan Trading AG, model: infinite® M200 )
Clean bench laminar air-flow-hood with burner (Harbin East Electronic Technology, model: HD-1360 )
Common plant tissue culture equipment (Measuring cylinder/volumetric flask, Beaker, Weighing machine, Autoclave, pH-meter, Scalpel, Fine forceps)
Software
ImageJ
Procedure
Ovary sterilization
Flowers (Figure 1) are collected with 1 to 3 cm anthocaulus from plants (the length of anthocaulus is usually 1 to 3 cm in our used cotton species, if some others is longer, you can cut as you wish; but the shorter ones is not recommended to choose unless there is no alternative). Remove the petals, stamens and bracts with hand carefully and thoroughly (or you can do it with some special accessory appliances but make sure do not hurt the cotton boll).
Figure 1. The organ of cotton flower
If the pistils are vigorous, such as those from in or before blooming flowers in which the pistils are linked with the cotton boll, while they will be physiological abscission after 2 day post anthesis (DPA), they should be kept to avoid liquid permeating into the cotton boll through the wound when sterilized.
Soak the whole remaining tissue (cotton boll linked with anthocaulus and/or pistils) into 0.1% (w/v) HgCl2 for 15 min immediately (occasional shaking/stirring is needed to keep all the tissue fully soaked), discard HgCl2 and rinse with sterilized ddH2O three times. Each for 2 to 5 min.
Ovules preparation
Hold the tissue with the anthocaulus, and remove the shuck of the ovary (or the young cotton boll) with a sterilized forceps carefully, then float and disperse the intact ovules on the liquid BT medium gently. Make sure the ovules are not injured, broken or attached the brown gossypol from the broken shuck.
For same batch culture, equal ovules should be put in each flask and usually less than 20 ovules for each batch.
If the ovules are going to culture for more than 10 days, the ovules sum in each flask should be less than 10 at the beginning.
For young ovules that have not grown fiber, they could be separated easily, while for ovules with longer fiber that are sticking together, such as the ovules in 2 day post anthesis (DPA), they need to be carefully separated one by one with forceps before floating on the medium.
Label the flask with the date, the age of the ovules and the hormones contained.
Ovule culture
The floating ovules are incubated in 30 °C without light and shake.
The fiber should be easily visible after 4-5 days of culture for the 0 DPA ovules (Figure 2).
Figure 2. 0 DPA ovules after 5 days culture
The ovules or fiber can be used for further analysis, such as fiber production measurement, RNA extraction and biochemical analysis etc.
Fiber measurement
There are two methods to measure fiber production.
Total fiber units (TFU) measurement, which was also innovated by Beasley et al. (1974).
For 12 days cultured ovules, 10 ovules for each assay. Ovules are dried out of medium with filter paper. Sunk in boiling water for 2 min, then dried again with filter paper for 2 to 3 min.
Stain the ovules in a small beaker with 20 ml 0.02% Toluidine blue O for 30 sec, then discard the liquid with a fine sieve and wash in running water for 1 min immediately to remove the non-absorbed dye.
Dry the ovules, then destain in flask with 20 ml glacial acetic acid-ethanol-water (10:95:5) for 2 h. The flask should be sealed to avoid liquid evaporation which can cause differences in concentration of destaining liquid.
Measure the absorbance of the destaining liquid in 624 nm and count as the relative yield of fiber.
Fiber length measurement (Figure 3)
Ovules are sunk in boiling water for 2 min or in 75% ethanol for 15 min, then put the ovules in slowly running water to make the fiber flow to one side and measured with a ruler.
Or put the ovules on a glass slide, comb the fiber gently with a dissecting needle, then take photos and measure the length with a ruler or you can use software for automation of the measurement (e.g. Image J).
Figure 3. Cultured ovule (1) is soaked in 75% ethanol (2) to separete fiber and measured on a glass slide (3)
Notes
This protocol can be used for analyzing the effects of dozen of chemicals, including hormones, plant growth regulators, plant growth inhibitors and flavonoids (Tan et al., 2013) etc., on fiber development in our lab. We found it functioned similarly in Gossypium hirsutum (more than 4 cultivars were applied), G. arboreum and G.herbaceum. But for G. barbadense, there will be several differences in the fiber growth rate and hormones response. The fiber will grow later and fewer, and GA3 can’t promote fiber grow for the 0 DPA ovules independently.
While it is a very sensitive system, many factors are involved in a success assay, especially for that of ovule developmental stage. According to our unpublished data, there are dramatic differences for 0 DPA ovules from 7 am to 12 am in response to IAA and GA3. And GA3 could promote fiber growth independently on the 0 DPA ovules post 12 am except for that from G. barbadense. The ovules before 0 DPA may be not easy to float on the liquid medium, should be taken more gently. And ovules of different developmental stages are also response differentially to chemical treatment (Tan et al., 2012). So for the same assay, the ovules should be from the same stage and all the manipulations should be strictly consistent and control should be set up for each. The samples from different batches could not analyze together.
CaCl2.2H2O should be separately prepared and stored with KH2PO4 and MgSO4.7H2O, be carefully mixed and diluted before use.
Other chemical should be sterilized first and added into the medium with IAA and/or GA3. Each new chemical should be undergone a dose test to determine the optimal dose.
Infection rate of ovules from the glasshouse is less than that from the field.
Take a simple plan before start. It will take a lot of time to release the ovules from the ovary (approximately 10 ovules per hour for freshman).
For TFU assay, it is 10 ovules for each assay usually, while it should be changed depending on the days of culture and the bulk of ovules.
Recipes
BT medium preparation
Preparation of 20x Macro element stock solution
(stored in 4 °C)
Chemicals (company)
Mol.wt
Final conc.
(mg/L)
Final conc.
(mM)
Conc. stock
(mM)
mg to take
Final volume of stock
KH2PO4
136.09
272.18
2
40
5,443.6
1,000 ml
CaCl2.2H2O
147.01
441.06
3
60
8,821.2
MgSO4.7H2O
246.47
493.00
2
40
9,860
KNO3
101.10
5,055.50
50
1,000
101,110
Preparation of 100x Micro element stock solution
(stored in 4 °C)
H3BO3
61.83
6.183
0.1
10
618.3
1,000 ml
Na2MoO4.2H2O
241.95
0.242
0.001
0.1
24.2
KI
166
0.830
0.005
0.5
83
CoCl2.6H2O
237.93
0.024
0.0001
0.01
2.4
MnSO4.H2O
169.02
16.902
0.1
10
1,690.2
ZnSO4.7H2O
287.56
8.627
0.03
3
862.7
CuSO4.5H2O
249.69
0.025
0.0001
0.01
2.5
Preparation of 100x Fe salt stock solution
(stored in brown bottle and 4 °C)
FeSO4.7H2O
278.01
8.341
0.03
3
834.1
1,000 ml
Na2EDTA
372.24
11.167
0.03
3
1,116.7
Preparation of 1,000x Vitamins mixture stock solution
(stored in 4 °C)
VB1
337.27
1.349
0.004
4
1,349
1,000 ml
VB6
205.64
0.822
0.004
4
822
VB3
123.11
0.492
0.004
4
492
Preparation of 100x Inositol stock solution
(stored in 4 °C)
Inositol
180.16
180.160
1
100
18,016
1,000 ml
Hormone preparing
The general hormones for cotton ovules culture are IAA and GA3.The stock solutions of IAA and GA3 are 5 mM and 0.5 mM, respectively. Both are 1,000x solutions, pre-dissolved in a small volume (500-1,000 μl) of 95% ethyl alcohol, then brought to volume with sterilized double-distilled H2O water, aliquoted into 1.5 ml microcentrifuge tubes, sealed and stored in -20 °C. Usually 10 to 50 ml stock solution is prepared for each time.
Working BT medium preparation
Stock
Conc. of stock
Amount of stock soln to take
Final volume of media
Macro-element mixture
20x
50 ml
1,000 ml
Micro-element mixtures
100x
10 ml
Fe salt solution
100x
10 ml
Vitamin mix
1,000x
1 ml
Inositol
100x
10 ml
Glucose
24 g
pH 5.0
Medium is prepared in 100, 200 ml or other certain volumes before used, after sterilized, added suitable hormones, mixed and aliquoted into sterilized 50 ml flasks with about 10 ml each. Label the flask with what kinds of hormones contained, preparation and expiration dates (the medium should be used within less than 15 days).
Acknowledgments
The protocol was based on Beasley and Ting’s original work (Beasley and Ting, 1973), and adapted from two of our previous published work (Tan et al., 2012; Tan et al., 2013). This work was supported by the University Scientific and Technological Self-innovation Foundation, the National Natural Science Foundation of China (grant no. 30871560 and 31230056) and the National High-Tech Program of China (grant no. 2012AA101108).
References
Beasley, C. and Ting, I. P. (1973). The effects of plant growth substances on in vitro fiber development from fertilized cotton ovules. Am J Bot 130-139.
Beasley, C. A., Birnbaum, E. H., Dugger, W. M. and Ting, I. P. (1974). A quantitative procedure for estimating cotton fiber growth. Stain Technol 49(2): 85-92.
Tan, J., Tu, L., Deng, F., Hu, H., Nie, Y. and Zhang, X. (2013). A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid naringenin. Plant Physiol 162(1): 86-95.
Tan, J., Tu, L., Deng, F., Wu, R. and Zhang, X. (2012). Exogenous jasmonic acid inhibits cotton fiber elongation. J Plant Growth Regul 31(4): 599-605.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Plant Science > Plant developmental biology > Morphogenesis
Plant Science > Plant physiology > Plant growth
Plant Science > Plant cell biology > Tissue analysis
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973 | https://bio-protocol.org/en/bpdetail?id=973&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Cell Cycle Analysis in the Vertebrate Brain Using Immunolabeled Fresh Cell Nuclei
Noelia Lopez-Sanchez
Jose M. Frade
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.973 Views: 16807
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Apr 2013
Abstract
Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol allows the discrimination of single nuclei from doublets and larger aggregates, obtaining low coefficients of variation for cell cycle analysis with propidium iodide. In addition, due to the reduced sample handling this method has high recovery and good reproducibility. As an example, in this protocol we describe the isolation of cell nuclei from adult cerebral cortex, which are subsequently immunostained with antibodies against NeuN (a general neuronal marker) and EGR1 (an early response gene expressed by functionally active neurons), and subjected to flow cytometric gating and analysis. Nevertheless, the protocol can also be applied to other neural tissues from adult and embryonic brain.
Keywords: Flow cytometry DNA content Ploidy Tetraploid neuron Diploid neuron
Materials and Reagents
Frozen tissue samples (adult mouse hemicortex)
Phosphate-buffered saline (PBS)
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Protease inhibitors cocktail tablets (cOmplete, Mini, EDTA-free) (Roche Diagnostics, catalog number: 11 836 170 001 )
Bovine serum albumin (BSA) (stock 30 mg/ml in PBS) (Sigma-Aldrich, catalog number: A4503 )
Calf serum (Life Technologies, catalog number: 16170-086 )
Primary antibodies:
Mouse Anti-NeuN antibody (clone A60) (EMD Millipore, catalog number: mab377 )
Rabbit anti-EGR1 antibody (clone T.126.1) (Thermo Fisher Scientific, catalog number: MA5-15009 )
Secondary antibodies:
Alexa Fluor 647 Goat Anti-Mouse IgG (H + L) Antibody (Life Technologies, catalog number: A21236 )
Alexa Fluor 488 Donkey Anti-Rabbit IgG (H + L) Antibody (Life Technologies, catalog number: A21206 )
Propidium iodide (stock 1 mg/ml, prepared in autoclaved water) (Sigma-Aldrich, catalog number: P4170 )
RNase A (stock 1 mg/ml, inactivated by boiling as indicated by the manufacturer) (Sigma-Aldrich, catalog number: R5000 )
DAPI (stock 100 μg/ml, prepared in autoclaved water) (Sigma-Aldrich, catalog number: D9542 )
Equipment
15 ml tube
1.5 ml Eppendorf tube
7 ml Dounce homogenizer (WHEATON, model: 357542 )
Microscope
Refrigerated centrifuge
Autoclaved 40 μm nylon filters
Emission filters: BP 530/30, BP 616/23, BP 660/20, APC, FITC
FACSAria cytometer (BD Biosciences) equipped with a double argon (488 nm) and helium-neon (633 nm) laser
Software
FACSDiva (BD Biosciences)
Modfit LT software (Verity Software)
Procedure
Cell nuclei isolation
Prepare homogenization buffer containing PBS, 0.1% Triton X-100, and one tablet of protease inhibitors cocktail per 10 ml of buffer.
Note: Keep buffers and sample on ice during processing.
Transfer one mouse hemicortex (or an equivalent mass of adult nervous tissue) to a Dounce homogenizer containing 3 ml of homogenization buffer. The tissue is then homogenized on ice with the “loose” and “tight” pestles, as indicated by the manufacturer.
Note: Volumes can be scaled according to sample size and complexity (for instance, 1 ml homogenization buffer for one telencephalic vesicle from an E17 embryo or one adult murine hippocampus). Several tissues can be processed in parallel, this will allow to get aliquots for control immunostainings (see step B2).
Collect the sample and add 1.5 ml of PBS-0.1%Triton X-100 (final volume 4.5 ml).
Centrifuge at 200 x g for 1.5 min at 4 °C to remove undissociated tissue (mostly blood vessels).
Optional: Here and in subsequent centrifugation steps the sample can be aliquoted in 1.5 ml Eppendorf tubes to improve the visualization of the pellets.
Discard pellet and transfer the supernatant to a 15 ml tube.
Note: At this step you can remove a small aliquot of the sample, which should be labeled for at least 1 min with either DAPI (100 ng/ml) or propidium iodide (25 μg/ml) to check it under the microscope (Figure 1, Homog.).
Add ice-cold PBS to a final volume of 12 ml.
Note: Volumes can be scaled according to sample size and tissue structure. For instance, for embryonic tissues the volume can be reduced due to their low complexity (one telencephalic vesicle from an E17 embryo can be washed in 1.5 ml PBS). For one adult hippocampus 5 ml PBS are required.
Centrifuge at 400 x g for 4 min at 4 °C.
Discard supernatant and carefully add PBS to the pellet (total volume of 1-1.2 ml).
Notes:
CRITICAL STEP: Do not disturb the pellet nor try to resuspend it at this point. It is crucial not to disturb the cell nuclei to maintain their integrity!
At this step you can remove a small aliquot of the supernatant and, after adding either DAPI (100 ng/ml) or propidium iodide (25 μg/ml), check under the microscope that cell nuclei are absent (Figure 1, Supernat.).
Maintain the cell nuclei pellet on ice for 20-30 min.
Resuspend the pellet by gently swirling of the vial (until no visible lumps can be observed), and then by pipetting up and down.
Notes:
At this step remove a small aliquot of the sample and, after adding either DAPI (100 ng/ml) or propidium iodide (25 μg/ml) check the quality of cell nuclei under the microscope (Figure 1, Sample).
Samples can be stored at 4 °C for a few hours until immunostaining and for 24 h for cell cycle analysis.
If immunostaining is not required proceed to step B6.
Figure 1. Microscopic images of the aliquots obtained during the cell nuclei isolation procedure. Aliquots of the samples obtained in steps A5 (Homog.), A8 (Supernat.), and A10 (Sample) stained with DAPI (blue).
Fresh/unfixed nuclei immunostaining
To block nonspecific immunostaining, add 4-5 μl calf serum and 6.4-8 μl BSA (30 mg/ml) to 400-500 μl of the cell nuclei suspension obtained in step A10. Mix by inversion and proceed without further delay.
Prepare three control samples (400-500 μl each) by pooling small aliquots of the cell nuclei suspensions obtained in step A10, and block nonspecific immunostaining as indicated above:
Secondary antibody control (CONT).
Positive control for NeuN (POSITIVE NEUN+).
Positive control for EGR1 (POSITIVE EGR1+).
Note: This allows to keep enough volume from the cell nuclei suspensions obtained in step A10 to perform at least two different immunostainings (even for small tissues such as the hippocampus).
Add primary antibody/antibodies against nuclear proteins and mix by inversion:
Secondary antibody control: No primary antibody.
Positive control for NeuN: 0.6 μl of mouse anti-NeuN in 500 μl of sample.
Positive control for EGR1: 1 μl of rabbit anti-EGR1 in 500 μl of sample.
Sample: 0.6 μl of mouse anti-NeuN and 1 μl of rabbit anti-EGR1 in 500 μl of sample.
Note: Dilutions for other primary antibodies should be empirically optimized (usually the same or double concentration as used for immunohistochemistry should be a good choice). If possible, select antibodies known to recognize unfixed/native epitopes (i.e. validated for immunoprecipitation).
Without washing, add 1/500 dilution of secondary antibody/antibodies to all the samples (including the controls) and mix by inversion.
Note: We combine primary and secondary antibodies to avoid repeated washing steps that could result in loss of material. Be sure that the fluorophores you use are both excitable and detectable by the cytometer and that they are compatible with the excitation/emission spectra of each other, as well as with that of propidium iodide.
Incubate over night at 4 °C in the dark (without shaking).
Note: Alternatively, samples can be incubated for 2 h at room temperature.
Carefully resuspend the sedimented nuclei and, without washing, filter the sample through a 40 μm autoclaved (i.e. DNase-free) nylon filter.
Note: At this step remove a small aliquot of the sample and, after adding either DAPI (100 ng/ml) or propidium iodide (25 μg/ml), check under the microscope the immunstaining signals (Figure 2).
Figure 2. Microscopic images of the aliquots of cell nuclei obtained after immunostaining
Add propidium iodide to the samples to a final concentration of 25-50 μg/ml and DNase-free RNase A to a final concentration of 25 μg/ml.
Flow cytometric gating and analysis
Analyze the samples using a flow cytometer.
Note: In our case, we use a FACSAria cytometer equipped with a double argon (488 nm) and helium-neon (633 nm) laser. Emission filters: BP 530/30 (for Alexa 488), BP 616/23 (for propidium iodide), and BP 660/20 (for Alexa 647). Data are collected using a linear digital signal process. For data analysis, we use FACSDiva and Modfit LT software.
Localize the nuclei population in the (forward scatter) FSC vs. (side scatter) SSC plot, and create a first gate: P1 (Figure 3A).
Simultaneously eliminate the debris (i.e. the population unstained for IP) by creating a P2 gate in the Propidium Iodide-A vs. Propidium Iodide-H plot (Figure 3B). This should greatly improve the identification of the nuclei in the FCS-A vs. SSC-A plot (Figure 3C), as well as the fine-tuning of P1 (Figure 3A).
Select the singlet population and exclude doublets and clumps by gating (P3) on the Propidium Iodide-A vs. Propidium Iodide-H plot (Figure 3D) (see Figure 4 for further explanation).
Figure 3. Contour plots and gated scheme used to detect nuclei from cerebral cortex of adult mice
Figure 4. Scheme explaining the procedure used for doublet discrimination. As propidium iodide-labeled nuclei transit through the laser beam, the fluorescence signal is converted into a voltage pulse defined by its height (H), width (W), and area (A). The integrated area of the signal (Propidium iodide-A) is proportional to the DNA content. The Propidium Iodide-W variable directly depends on the time spent by the particle crossing the laser beam and the Propidium Iodide-H reflects the maximal intensity of fluorescence signal. A 4C singlet (tetraploid nucleus) can be discriminated from a doublet (two diploid nuclei adhered to each other) due to the higher H/A ratio and lower W/A ratio of its fluorescence signal (adapted from López-Sánchez and Frade, 2013).
Confirm the correct exclusion of doublets in the selected population by checking the Propidium Iodide-A vs. Propidium Iodide-W plot and adjust P3 if needed. The population with 4C DNA content and high Propidium iodide-W should disappear (compare Figure 3E with Figure 3F). The resulted population is constituted by the singlets to be analyzed henceforth.
In the control sample (CONT), determine the background threshold of fluorescence signals using emission filter APC (for NeuN) and emission filter FITC (for EGR1). The threshold can be determined representing individual signals in a histogram (Figure 5A-B) or simultaneously representing both signals on the same dot plot (Figure 6A).
Figure 5. Histograms representing the fluorescence signals for NeuN and EGR1 in secondary antibody controls (CONT) for NeuN (A) and EGR1 (B), as well as sample populations (SAMPLE) immunostained for NeuN (C) and EGR1 (D)
Figure 6. Dot plots representing the fluorescence signals for NeuN and EGR1 in secondary antibody control (A), positive control for NeuN (B), positive control for EGR1 (C), and sample populations (D)
Select positive populations in the individual positive controls (Figure 5C-D and Figure 6B-C). This allows to identify the double-labeled population (Figure 6D).
Note: Sometimes nuclei of interest have specific characteristics of size or complexity that can be used for defining specific nuclei populations. For instance, most of the NeuN positive nuclei from adult mice has higher SSC-A than the negative ones (Figure 7). This allows to improve the correct gating of this population.
Figure 7. Dot plots of NeuN vs. SSC-A signals
Finally, analyze the DNA content of the population/s of interest in each sample.
Note: This method is useful for both cell cycle analysis in proliferating tissues (Figure 8A-B) and quantification of DNA content in NeuN+ nuclei (Figure 8C-D).
Figure 8. DNA content analysis. Typical histogram of DNA content from a proliferating tissue (A), and analysis of its cell cycle phases quantified with Modfit software (B). Representative histograms showing non-neuronal (C) and neuronal (D) cell nuclei from adult mouse brain. 2C: diploid, 4C: tetraploid.
Notes
Similar results have been obtained with nervous tissue from other vertebrates, including embryonic and post hatch chicken (Lopez-Sanchez and Frade, 2013) and human (unpublished data).
Acknowledgments
This protocol is adapted from a previous paper by Lopez-Sanchez and Frade (2013). The experimental work was supported by grants from the “Ministerio de Ciencia e Innovación” (BFU2009-07671 and SAF2012-38316) and “Fundación Areces” (CIVP16A1815). Noelia López-Sánchez acknowledges a JAE-Doc contract (JAEDoc026, 2008 call) from the CSIC program “Junta para la Ampliación de Estudios”, co-funded by European Social Fund.
References
López-Sánchez, N. and Frade, J. M. (2013). Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice. J Neurosci 33(17): 7488-7500.
Article Information
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© 2013 The Authors; exclusive licensee Bio-protocol LLC.
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Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Lopez-Sanchez, N. and Frade, J. M. (2013). Cell Cycle Analysis in the Vertebrate Brain Using Immunolabeled Fresh Cell Nuclei. Bio-protocol 3(22): e973. DOI: 10.21769/BioProtoc.973.
López-Sánchez, N. and Frade, J. M. (2013). Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice. J Neurosci 33(17): 7488-7500.
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Category
Neuroscience > Cellular mechanisms
Cell Biology > Cell-based analysis > Flow cytometry
Cell Biology > Organelle isolation > Nuclei
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974 | https://bio-protocol.org/en/bpdetail?id=974&type=0 | # Bio-Protocol Content
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Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry
AL Ana Luque
SC Sonia Cárdenas-Brito
RO Rut Olivar
Josep M. Aran
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.974 Views: 17463
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Original Research Article:
The authors used this protocol in The Journal of Immunology Mar 2013
Abstract
Antigen uptake by dendritic cells is the first key step towards induction of antigen-specific T-cell responses. This flow cytometry-based protocol describes the analysis of dendritic cell uptake of soluble antigens through two different mechanisms: non-specific macropinocytosis (using Lucifer Yelloy CH), and receptor-mediated endocytosis (using DQTM Ovalbumin). The protocol is generated based on data presented in Olivar et al. (2013).
Keywords: Dendritic cells Endocytosis Flow cytometry Fluorescent dye
Materials and Reagents
Whole blood
RPMI 1640 Medium, GlutaMAXTM (Gibco®, catalog number: 61870 )
DPBS without Ca2+ and Mg2+ (Gibco®, catalog number: 14190-169 )
100x liquid Penicillin-Streptomycin (Gibco®, catalog number: 15140-122 )
200 mM L-Glutamine solution (Gibco®, catalog number: 25030-024 )
Fetal Bovine Serum (FBS) (Gibco®, catalog number: 10270106 )
Lipopolysaccharide from Escherichia coli 026:B6 (10 mg) (Sigma-Aldrich, catalog number: L2654 )
Lucifer Yellow CH dilithium salt (25 mg) (Sigma-Aldrich, catalog number: L0259 )
DQTM Ovalbumin (1 mg) (Molecular Probes®, catalog number: D-12053 )
Ficoll-Paque PLUS (General Electric Company, catalog number: 17-1440-03 )
GMP Recombinant Human Interleukin-4 (50 μg, 13 x 106 IU/mg) (Gentaur Molecular Products, catalog number: 04-GMPhuIL4-50 μg )
Recombinant Human GM-CSF (300 μg, 3.88 x 106 IU/vial) (Gentaur Molecular Products, catalog number: 04- RHUGM-CSF-300 μg )
IL-4
Bovine Serum Albumin Fraction V (BSA) (Roche Diagnostics, catalog number: 10735078001 )
FITC-conjugated anti-CD14 (RMO52) (Beckman Coulter, catalog number: IM0645U )
FITC-conjugated anti-IgG2a (7T4-1F5) (Beckman Coulter, catalog number: IM0645U)
Perfect-Count MicrospheresTM (Cytognos S. L., catalog number: CYT-PCM-50 )
NaN3 (Sigma-Aldrich, catalog number: 71289 )
FACS buffer (see Recipes)
Complete medium (see Recipes)
DQ-OVA (1 mg/ml) (see Recipes)
Lucifer Yellow (10 mg/ml) (see Recipes)
rHuIL-4 (500 IU/ml) (see Recipes)
rHuGM-CSF (800 IU/ml) (see Recipes)
LPS (1 mg/ml) (see Recipes)
Equipment
15 ml Ficoll-Paque PLUS
60-mm cell culture plates (Corning, catalog number: 15 430166 )
Cytometer tubes (BD Falcon tubes, round-bottom) (Becton, Dickinson and Company, catalog number: 352052 )
Centrifuge Heraeus Multifuge 3 L-R (Heraeus Holding, catalog number: 75004370 )
37 °C, 5% CO2 cell culture incubator
BD FACSCalibur flow cytometer (Becton, Dickinson and Company, catalog number: 342975 )
Software
CellQuest Pro software (Becton, Dickinson and Company, catalog number: 643436 )
Procedure
Dilute 25 ml of buffy coat (initial leukocyte concentrate from a whole blood donation) with the same volume of DPBS.
Prepare two 50 ml tubes with 15 ml Ficoll-Paque PLUS. Carefully layer 25 ml of the diluted blood sample on Ficoll-Paque PLUS. Important: when layering the sample do not mix Ficoll-Paque PLUS and the diluted blood sample.
Centrifuge at 400 x g for 25 min at 18-20 °C. Important: brakes off.
Soak up the white interphase between the diluted plasma fraction and the transparent ficoll fraction with a pipette and transfer it into a fresh tube.
Wash twice with DPBS.
Resuspend the pellet in 5 ml DPBS.
In a cytometer tube mix 3 μl of FITC-conjugated anti-CD14 antibody plus 60 μl DPBS and 20 μl of cellular suspension.
Incubate 15-18 min at room temperature.
Add 120 μl DPBS and count the number of CD14+ monocytes by flow cytometry using Perfect-Count MicrospheresTM according to the manufacturer’s instructions.
Plate monocytes at 1 x 106 cells/ml in 60-mm culture plates, in RPMI 1640 medium without serum, and allow to adhere for 2 h at 37 °C in 5% CO2.
Remove the non-adherent cells by washing in DPBS. The final population of adherent cells contains 75-80% of monocytes, as demonstrated by flow cytometry of anti-CD14–stained isolates.
Generate monocyte-derived DCs by supplementing the monocyte cultures with 1 ml of complete RPMI 1640 medium plus GM-CSF (800 IU/ml) and IL-4 (500 IU/ml).
At day 3 add 1ml of complete RPMI 1640 medium plus GM-CSF (800 IU/ml) and IL-4 (500 IU/ml).
For DC maturation, at day 5 replace the old medium with fresh complete RPMI 1640 medium plus GM-CSF (800 IU/ml) and IL-4 (500 IU/ml) and stimulate the immature DCs for 48 h with 5 μg/ml LPS.
Harvest the non-adherent cells floating in the culture medium in a 15 ml tube (at day 5 for immature DCs; at day 7 for mature DCs). Add 2 ml DBPS (37 °C), rinse and collect the adhered cells by pipetting. Wash twice more with DPBS and pool both floating and adherent cells. Centrifugue and resuspend the pellet in 500 μl of complete medium.
Prepare two cytometer tubes with 60 μl of complete medium plus 4 μl DQ-OVA (stock: 1 mg/ml) at 37 °C or 0 °C.
Prepare two cytometer tubes with 60 μl of complete medium plus 6 μl Lucifer Yellow CH (stock: 10 mg/ml) at 37 °C or 0 °C.
Add 100 μl of cell suspension (~ 2 x 105 cells/ml) to each cytometer tube.
Incubation time: 15 min for DQ-OVA; 120 min for Lucifer Yellow CH. The fluorescence of OVA labeled with BODIPY FL dye (DQ-OVA) is self-quenched until the OVA is taken up via the mannose receptor and degraded only by endolysosomal proteases. Lucifer Yellow CH (LY) is a hydrophilic tracer for fluid-phase macropinocytosis. LY is not degraded and is nontoxic at concentrations up to 6 mg/ml.
Stop the incubations by adding 1 ml cold FACS buffer.
Wash the cells two times with cold FACS buffer.
Analyze the incorporated fluorescence of both immature DCs (Figure 1) and mature DCs using flow cytometry. Compare the histograms and corresponding mean fluorescence intensities (MFI) between cells incubated at 37 °C (specific uptake) and cells incubated at 0 °C (non-specific uptake: passive diffusion,…).
Figure 1. Analysis of the endocytic activity of immature DCs by flow cytometry. The endocytic activity of monocyte-derived immature DCs was assessed measuring the uptake of the fluorescent reporters DQ-OVA (receptor-mediated endocytosis) and Lucifer Yellow CH (fluid-phase endocytosis). Representative histograms are shown. Dye uptake controls are displayed in gray. The median fluorescence intensities (MFI) for the different fluorescent cell populations are indicated in each histogram.
Recipes
FACS buffer (500 ml)
Mix 5 g BSA and 0.5 g NaN3 with 500 ml 1x DPBS
Filter sterilize (0.45 μm)
Stored at 4 °C
Complete medium
RPMI 1640 medium, GlutaMAXTM
100 μg/ml streptomycin
100 IU/ml penicillin
2 mM L-glutamine
10% heat-inactivated FBS
GM-CSF 800 IU/ml
IL-4 500 IU/ml
Stored at 4 °C
DQ-OVA (1 mg/ml)
A 1 mg/ml solution can be prepared by dissolving the contents of one vial in 1 ml of DPBS. Once reconstituted, the solution should be stored at -20 °C, protected from light.
Lucifer Yellow (10 mg/ml)
A 10 mg/ml solution can be prepared by dissolving the contents of one vial in 2.5 ml of dH2O. Once reconstituted, the solution should be stored at 4 °C, protected from light.
rHuIL-4 (500 IU/ml)
A 500 IU/ml solution can be prepared by dissolving the contents of one vial in 500 μl of dH2O. Once reconstituted, the solution should be stored at -80 °C.
rHuGM-CSF (800 IU/ml)
A 800 IU/ml solution can be prepared by dissolving the contents of one vial in 2 ml of dH2O. Once reconstituted, the solution should be stored at -80 °C.
LPS (1 mg/ml)
A 1 mg/ml solution can be prepared by dissolving the contents of one vial in 1 ml of DPBS. Once reconstituted, the solution should be stored at -20 °C.
Acknowledgments
This protocol was adapted from the previously published study, Olivar et al. (2013), and was supported by the Ministerio de Ciencia e Innovación (Madrid, Spain), through grant PI10/1073 from the “Fondo de Investigaciones Sanitarias” (FIS-ISCIII), and from 2009SGR1490 (Generalitat de Catalunya) to JMA. JMA is sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES06/012).
References
Olivar, R., Luque, A., Naranjo-Gomez, M., Quer, J., Garcia de Frutos, P., Borras, F. E., Rodriguez de Cordoba, S., Blom, A. M. and Aran, J. M. (2013). The α7β0 isoform of the complement regulator C4b-binding protein induces a semimature, anti-inflammatory state in dendritic cells. J Immunol 190(6): 2857-2872.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Luque, A., Cárdenas-Brito, S., Olivar, R. and Aran, J. M. (2013). Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry. Bio-protocol 3(22): e974. DOI: 10.21769/BioProtoc.974.
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Category
Immunology > Immune cell function > Dendritic cell
Cell Biology > Cell-based analysis > Flow cytometry
Cell Biology > Cell-based analysis > Cytosis > Endocytocis
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975 | https://bio-protocol.org/en/bpdetail?id=975&type=0 | # Bio-Protocol Content
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Peer-reviewed
Shigella IpaD and IpaB Surface Localizations
Lionel Schiavolin
AM Alaeddine Meghraoui
AA Abdelmounnaim Allaoui
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.975 Views: 10249
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Microbiology Apr 2013
Abstract
Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).
Keywords: Type 3 secretion system Tip complex Shigella Flow cytometry
Materials and Reagents
Shigella strains
Tryptic Soy Broth (TSB) (VWR International, catalog number: for Europe 1.00525.5000 and for U.S. EM1.00525.5007 )
Agar (MP Biomedicals, catalog number: 0 210026291 )
Congo Red (VWR International, catalog number: for Europe 34140.184 )
Anti-IpaD and -IpaB polyclonal antibodies (house made)
PBS (Fischer Scientific, catalog number: BP399-20 )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
Triton X-100 (VWR International, for Europe catalog number: 1.08603.1000 )
Trizmabase (Sigma-Aldrich, catalog number: T1503 )
Bovine serum albumin (BSA) (VWR International, catalog number: 422361V )
Anti-mouse secondary antibody CF647-conjugated (Sigma-Aldrich, catalog number: SAB4600351 )
PBS + PFA 4% stock solution (see Recipes)
Congo Red agar plates (see Recipes)
Equipment
250 ml Erlenmeyer
Microtubes
CR agar plate
Polystyrene tube for FACS analysis
Centrifuge Heating magnetic stirrer
37 °C shaker
Rotator mixer
Flow cytometry with a four-colour FACS Calibur cytometer (Becton Dickinson and Company)
Procedure
Preparation of bacterial samples
Launch overnight precultures in TSB (37 °C with shaking) from Congo Red (CR) positives colonies of Shigella on CR agar plates.
Dilute 1: 100 precultures in fresh TSB (for volume see step A3) and incubate at 37 °C with shaking until an OD600 ≈ 1.5 is reached. (Medium must be filtered or autoclaved with stirring to avoid glucose caramelization which interferes with type III secretion.)
Harvest 2 x 108 bacteria per tested conditions at 2,000 x g for 4 min at room temperature (RT) (OD600 of 1 correspond to approximately 5 x 108 bacteria).
Wash twice with 500 μl of ice-cold PBS with 0.1% Triton X-100 (centrifugation conditions as in step A3). Be careful when you take out the liquid supernatant as the pellet can detach gradually all along the procedure.
Resuspend in 500 μl of ice-cold PBS with 0.1% Triton X-100 and add 500 μl of PBS + PFA (4%) to fix bacteria.
Mix by inversion and incubate 20 min at RT.
Add 50 μl Tris HCl 1 M (pH 7.5), mix by inversion and incubate 5 min at RT to quench the cross-linker.
Harvest bacteria at 10,000 x g for 2 min at RT (All further centrifugation steps are performed with these parameters.).
Wash once with 1 ml PBS + 0.1% Triton X-100 and once with 1 ml PBS.
Immunological staining
Harvest bacteria and resuspend in 500 μl of the blocking solution (PBS + 4% BSA).
Incubate bacteria on rotator mixer 1 h at 4 °C.
Harvest bacteria and resuspend in 250 μl PBS + 4% BSA with mouse sera diluted 1: 100.
Sera were from Swiss mice immunized with GST-IpaD131-332 (Schiavolin et al., 2013) or His-IpgC + IpaB (Page et al., 1999). Negative controls for IpaD and IpaB localizations are respectively an IpaD protein lacking its last residues which do not bind the needle tip (Espina et al., 2006) and an ipaD KO mutant where IpaB is not retained at the tip (Veenendaal et al., 2007). In both cases secreted IpaD or IpaB do not interfere with the assay.
Incubate overnight at 4 °C with agitation.
Add 750 μl PBS to bacteria and then wash twice with 1 ml PBS. All further steps are made in the dark to preserve fluorescent properties of the secondary antibody. Microtubes or rack are covered with aluminum foil.
Resuspend in 250 μl of PBS + 4% BSA with goat CF647-conjugated anti-mouse IgG antibody diluted 1: 500.
Agitate 1 h or longer at 4 °C (incubation time may last overnight).
Add 750 μl PBS to bacteria and then wash twice with 1 ml PBS.
Resuspend bacterial pellet in 500 μl of PBS and dilute 1: 10 in FACS polystyrene tube.
Analyze by flow cytometry with a four-colour FACS Calibur cytometer for instance (you will find examples of results in the supporting information of the third reference (Schiavolin et al., 2013) available for free on Mol. Microbiol. website).
Parameters used (no compensation):
detectors
voltage
mode
FSC
E02
Log
SSC
366
Log
FL4
800
Log
Recipes
PBS + PFA 4% stock solution (100 ml)
Weigh out 4 g of paraformaldehyde in a 250 ml erlenmeyer
Add 80 ml of double-distilled water
Add 50 μl of 1 M NaOH
Add a magnetic bar and close the erlenmeyer
Heat at 70 °C until complete solubilization (the solution should become clear)
Put on ice and allow the solution to cool down to RT
Adjust volume to 90 ml with double-distilled water
Add 10 ml of PBS 10x and mix
Filter solution with a 0.2 μm 25 mm nylon syringe filter and aliquot in 15 ml falcon tube
Freshly prepared PFA can be stored at -20 °C for further assays (thaw gently at RT)
Congo Red agar plates
Prepare a 30 g/L TSB solution with bidistilled water and dissolve 15 g/L agar
Autoclave the medium for 15 min at 120 °C (If you autoclave your medium for 20 min, sugar will caramelize and your plates will be darker, and even darkest after incubation with bacteria at 37 °C.)
Add the Congo Red at a final concentration of 250 µg/ml (Stock solution is prepared in bidistilled water at a concentration of 10 mg/ml.) when the bottle can be safely handled with a protective glove.
Acknowledgments
This protocol was adapted from Lee et al. (2010). This study was supported by grants from the Belgian Fonds National de la Recherche Scientifique Médicale (FRS-FNRS; Convention 3.4556.11) and from the European Community’s Seventh framework program FP7/2011–2015 under grant agreement No. 261742. L.S. and A.M are recipients of a PhD fellowship from the Belgian Fonds National de Recherches Industrielles et Agronomiques (FRIA). A part of this work was also supported by the Fonds Defay, and by Van Buuren and Héger-Masson foundations. We thank A. Op de Beeck for her help in performing the FACS experiment.
References
Espina, M., Olive, A. J., Kenjale, R., Moore, D. S., Ausar, S. F., Kaminski, R. W., Oaks, E. V., Middaugh, C. R., Picking, W. D. and Picking, W. L. (2006). IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect Immun 74(8): 4391-4400.
Lee, P. C., Stopford, C. M., Svenson, A. G. and Rietsch, A. (2010). Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV. Mol Microbiol 75(4): 924-941.
Page, A. L., Ohayon, H., Sansonetti, P. J. and Parsot, C. (1999). The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri. Cell Microbiol 1(2): 183-193.
Schiavolin, L., Meghraoui, A., Cherradi, Y., Biskri, L., Botteaux, A. and Allaoui, A. (2013). Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact. Mol Microbiol 88(2): 268-282.
Veenendaal, A. K., Hodgkinson, J. L., Schwarzer, L., Stabat, D., Zenk, S. F. and Blocker, A. J. (2007). The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol 63(6): 1719-1730.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Schiavolin, L., Meghraoui, A. and Allaoui, A. (2013). Shigella IpaD and IpaB Surface Localizations. Bio-protocol 3(22): e975. DOI: 10.21769/BioProtoc.975.
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Category
Microbiology > Microbial biochemistry > Protein > Immunodetection
Microbiology > Microbe-host interactions > Bacterium
Cell Biology > Cell imaging > Fluorescence
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976 | https://bio-protocol.org/en/bpdetail?id=976&type=0 | # Bio-Protocol Content
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Harvest and Culture of Mouse Peritoneal Macrophages
Mingfang Lu
AV Alan W. Varley
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.976 Views: 38212
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS Pathogens May 2013
Abstract
Peritoneal macrophages are used as primary macrophages in lots of studies, mainly because they are easy to obtain. Injection of thioglycollate broth i.p. induces inflammatory responses and elicits large numbers of macrophages. This protocol can be used for harvesting resident or thioglycollate-elicited peritoneal cells. Peritoneal macrophages are non-adherent in situ and when they are cultured in dishes, they become adherent so that macrophages may be separated from other types of cells in peritoneal cavity.
Materials and Reagents
C57BL/6 mouse
70% alcohol or isopropanol
PBS (Life Technologies, catalog number: 10010023 )
EDTA (Life Technologies, catalog number: 15575-020 )
RPMI 1640 (Cellgro®, catalog number: 10-041-CV )
10% heat-inactivated FBS (endotoxin < 0.06 EU/ml) (Hyclone, catalog number: SH30071.03HI )
Penicillin-Streptomycin-Glutamine (Life Technologies, catalog number: 10378016 )
Nonessential amino acids (Life Technologies, catalog number: 11140050 )
Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 )
Hepes (suitable for cell culture) (Sigma-Aldrich, catalog number: H0887 )
2-mercaptoethanol (EMD Millipore, catalog number: ES-007-E )
Thioglycollate medium (BD Biosciences, catalog number: 211716 )
Pyrogen-free water (Hyclone, catalog number: SH30529.03 )
cRPMI medium (see Recipes)
Equipment
5 ml syringe
12-well plate
15 ml sterile Falcon tubes
Needles (20 G and 25 G)
Centrifuge
37 °C, 5% CO2 cell culture incubator
300 ml flask with an aluminum foil lid
Laboratory oven
Procedure
Harvest and culture of resident peritoneal cells
Fill 5 ml of PBS with 5 mM EDTA in a 5 ml syringe with a 25 G needle.
Anesthetize and sacrifice mouse with CO2.
Place mouse with abdomen up on paper towel in hood.
Swab abdomen with 70% alcohol or isopropanol.
Make a small incision in the center of the skin overlying the peritoneal wall.
Note: Small incision is made on the skin to peel the skin off. Injection and extraction can be at different sites on peritoneal membrane.
Firmly pull skin to expose the peritoneal wall.
Insert the needle to peritoneal membrane; avoid inserting needle into guts or bladder. Inject 5 ml of PBS EDTA into peritoneal cavity.
Massage abdomen for approximately 10-15 sec.
Withdraw the needle slowly. Change the 25 G needle on the syringe to a 20 G needle.
Use one hand to push the fluid to one side of the peritoneum. Using the other hand, insert needle to the side of cavity with plenty of fluid and withdraw the fluid from peritoneum. Avoid fat, gut or mesentery, which may clog the needle. Try to draw as much fluid as possible. Usually, approximately 4 - 4.5 ml fluid can be recovered from one mouse.
Remove needle from syringe and dispense contents into a centrifuge tube on ice.
Centrifuge peritoneal cells (300 x g; 3 min) and collect cell pellet. Usually about 2-4 million resident peritoneal cells can be recovered from one C57BL/6 mouse using this method and about 50% are peritoneal macrophages.
Resuspend cell pellet from one mouse in 1 ml of cRPMI medium, count cells.
Culture peritoneal cells in a 12-well plate, 2 million/well in 1 ml cRPMI at 37 °C with 5% CO2 for 6-18 h. During this time, peritoneal macrophages adhere to the plastic surface. The floating non-macrophages can then be washed away by adding and aspirating 0.5 ml cRPMI medium twice.
The adherent macrophages are ready to use.
Harvest and culture thioglycollate-elicited peritoneal cells
Thioglycollate elicited peritoneal macrophages can be harvested and cultured in the same way as described in steps 1-15 with additional steps as shown below.
Preparation of 3% thioglycollate medium.
Heat a 300 ml flask with an aluminum foil lid (180-200 °C) in a laboratory oven for at least 18 h to get rid of endotoxin.
Suspend 6 grams of thioglycollate medium in 200 ml of pyrogen-free water.
Autoclave (15 psi/121 °C/15 min).
After cooling, aliquot to 15 ml sterile Falcon tubes. Store in a dark place at room temperature for 2 months before using. We have found that thioglycollate medium stored at room temperature for up to 2 years can still be used.
Inject 1 ml of aged thioglycollate i.p. per mouse. Wait for 4-5 days, harvest peritoneal cells. About 10 million macrophages can be recovered from one mouse.
Note: The study was performed under an IACUC approved protocol (LCID 11E).
Recipes
cRPMI medium
RPMI 1640 with 10% heat-inactivated FBS (endotoxin < 0.06 EU/ml)
2 mM L-glutamine
100 μM of nonessential amino acids
100 U/ml penicillin
0.1 mg/ml streptomycin
10 μM of sodium pyruvate
25 mM Hepes, pH 7.4
50 μM 2-mercaptoethanol
Acknowledgments
This research was supported by the Intramural Research Program of the NIH, NIAID
References
Lu, M., Varley, A. W. and Munford, R. S. (2013). Persistently active microbial molecules prolong innate immune tolerance in vivo. PLoS Pathog 9(5): e1003339.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Lu, M. and Varley, A. W. (2013). Harvest and Culture of Mouse Peritoneal Macrophages. Bio-protocol 3(22): e976. DOI: 10.21769/BioProtoc.976.
Lu, M., Varley, A. W. and Munford, R. S. (2013). Persistently active microbial molecules prolong innate immune tolerance in vivo. PLoS Pathog 9(5): e1003339.
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Category
Cell Biology > Cell isolation and culture > Cell isolation
Immunology > Immune cell isolation > Macrophage
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977 | https://bio-protocol.org/en/bpdetail?id=977&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Assay to Evaluate Vascular Permeability Induction in Mice
HP Henry Puerta-Guardo
AR Arturo Raya-Sandino
LG Lorenza González-Mariscal
VR Victor H. Rosales
JA José Ayala-Dávila
BC Bibiana Chávez-Mungía
DM Daniel Martínez-Fong
FM Fernando Medina
Juan E. Ludert
Rosa María del Angel
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.977 Views: 21859
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Original Research Article:
The authors used this protocol in Journal of Virology Jul 2013
Abstract
Dengue virus infection usually courses as a benign self-limited fever, called dengue fever. However, on occasions it can progress to a life-threatening complication known as severe dengue (SD). A hallmark of SD is a sharp increase in vascular permeability. Secondary infections are considered a risk factor to develop SD, presumably through a mechanism called Antibody-Dependent Enhancement (ADE) of infection in cells with the capacity to bind antigen-antibody complexes, such as macrophages, and to trigger a subsequent aberrant cytokine response. The massive release of cytokine from macrophages has been postulated to cause changes in vascular permeability. The vascular permeability assay presented in this protocol is designed to assess whether any compound or cell-secreted product or soluble factor present in sera from patients may induce plasma leakage in mice. This test was used in the laboratory to determine whether cytokines and soluble factors produced in vitro by macrophages infected with dengue virus or dengue virus in the presence of facilitating antibodies are able to induce plasma leakage in vivo. Macrophages were infected with dengue virus or dengue virus in the presence of facilitating antibodies for 48 h. After this time, the conditioned supernatant containing cytokines and soluble factors released by the macrophages were collected and inoculated intraperitoneally into CD-1 mice. Twenty four hours after the first inoculation, mice were reinoculated with a second dose with Evans blue dye. After another 24 h, mice were euthanized and the amount of Evans blue present in the blood and lung was determined by spectrophotometric analysis. The assay was able to show differences in the capacity of the conditioned media to induce vascular permeability changes in the inoculated animals (Puerta-Guardo et al., 2013).
Keywords: Dengue Antibody dependent enhancement Cytokines Tight junctions Plasma leakage
Materials and Reagents
Mice of six to seven weeks old (CD-1® Mouse, Crl:CD1 (ICR)) (Charles River Laboratories)
Evans Blue Dye (Sigma-Aldrich, catalog number: E2129 )
Ketamine (Sigma-Aldich, catalog number: K2753 )
Xylazine (Sigma-Aldich, catalog number: X1251 )
TNF-α (BD CBA flex set) (BD Biosciences, catalog number: 558273 )
10% Formalin (Sigma-Aldich, catalog number: HT501128 )
Formamide (Sigma-Aldich, catalog number: F8775 )
NaCl
KCl
Na2HPO4
KH2PO4
HCl
1x PBS (see Recipes)
1% (w/v) Evans blue dye solution in PBS (see Recipes)
Equipment
EDTA tubes
Lyophilizer
Centrifuge (Beckman Coulter, model: Allegra X-12R ) (Rotor SX4750)
Spectrophotometry (BioTek Instruments, model: ELx808 Absorbance Microplate Reader)
Procedure
Mice of six to seven weeks old and 18-20 g in weight in a minimal amount of 4 mice per group, were injected intraperitoneally with 2 doses (100 μl each) 24 h apart, of media or supernatants to be evaluated.
Since TNF-α has been described as a plasma leakage inductor, this cytokine can be used for the injection as positive control (4 ng/ml).
Together with the second dose of conditioned supernatant, mice were injected intraperitoneally with 4 ml/kg of weight a 1% Evans blue dye solution (w/v in PBS) allowing circulation for 24 h as was described previously (Manaenko et al., 2011).
Before samples were collected, mice were injected with anesthetics: ketamine (70 mg/kg) and xylazine (6 mg/kg), afterward blood samples were collected by cardiac puncture.
Blood was immediately placed in EDTA tubes.
Samples were diluted 1:2 v/v in PBS and plasma was separated by centrifugation at 3,000 rpm for 8 min.
The amount of Evans blue dye present in plasma was measured by spectrophotometry at 630 nm. Quantification was performed by comparison with a standard curve.
The standard curve was constructed using dilution of Evans blue dye in 1x PBS in a range from 3 to 400 ng/ml (R = 0.999).
To evaluate the pulmonary capillary leakage, the mice were perfused transcardially with 50 ml of PBS followed by 20 ml of 10% Formalin for tissue fixation. The lung tissues were removed and preserved in formalin solution at 4 °C until analysis.
The right lung was vacuum-dried, weight and frozen instantly in liquid nitrogen.
Evans blue dye was extracted from the lung by incubation at 65 °C with formamide (2 ml/g tissue) overnight (Peng et al., 2004).
The lung tissue was pelleted by centrifugation (12,000 x g for 30 min), and the concentration of Evans blue dye extracted in the supernatant was determined spectrophotometrically at 630 nm against a standard curve. Concentration of Evans blue dye was determined in ng/ml of lung tissue. However, this study was designed to compare the permeability induction capacity of different cell supernatants, thus, results were expressed as fold increases in relation to the mock or control condition. Nevertheless, results can also be expressed directly in ng/ml of tissue. Any statistically significant increase between the control and the experimental conditions is considered as leakage induction (Figure 1).
Note: This type of studies has to be conducted in accordance with official guidelines for the Standard Production, Care and Use of Laboratory Animals of each country.
Figure 1. Effect of conditioned supernatants on vascular permeability in vivo. Six to eight weeks old mice (CD1 strain) were injected twice intraperitoneally, 24 h apart, with conditioned supernatans. A. Solution of 1% Evans Blue dye (EVD) was injected together with the second dose. Twenty four hours later, EVD was extracted from plasma and lungs and quantified against standard curves. Values are expressed as ng/ml per plasma (panel A) or as fold increases per lung in relation to the control (panel B). Panels C and D, standard curves for EVD extracted from plasma and lungs, respectively. Four mice were included per condition. Statistical significance was *p ＜ 0.05 and **p ＜ 0.001. Mock: supernatant collected from mock infected cells. DENV: Supernatant collected from cells infected directly with dengue virus. DENV + Enh: Supernatant collected from cells infected in the presence of enhancing antibodies. DENV + Mut: Supernatant collected from cells infected in the presence of mutated antibodies (incapable of inducing enhancing). DENV+Neu: Supernatant collected from cells infected in the presence of neutralizing antibodies. TNF-α: TNF-α used as positive control.
Recipes
1x PBS
8 g (137 mM) NaCl
0.2 g (2.7 mM) KCl
1.44 g (10 mM) Na2HPO4
0.24 g (2 mM) KH2PO4
Add 800 ml H2O
Adjust pH to 7.4 with HCl
Add water up to 1 L
1% (w/v) Evans blue dye solution in PBS
Add 1 g of Evans blue dye to 100 ml of 1x PBS
Acknowledgments
This protocol was adapted from Puerta-Guardo et al. (2013). The study was partially supported by grants 103783 and 127447 from The Mexican Council for Science and Technology (CONACYT) to JEL and RMDA, respectively. HPG and ARS are recipients of CONACYT scholarships. Authors declare no conflict of interest.
References
Manaenko, A., Chen, H., Zhang, J. H. and Tang, J. (2011). Comparison of different preclinical models of intracerebral hemorrhage. Acta Neurochir Suppl 111: 9-14.
Peng, X., Hassoun, P. M., Sammani, S., McVerry, B. J., Burne, M. J., Rabb, H., Pearse, D., Tuder, R. M. and Garcia, J. G. (2004). Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Respir Crit Care Med 169(11): 1245-1251.
Puerta-Guardo, H., Raya-Sandino, A., Gonzalez-Mariscal, L., Rosales, V. H., Ayala-Davila, J., Chavez-Mungia, B., Martinez-Fong, D., Medina, F., Ludert, J. E. and del Angel, R. M. (2013). The cytokine response of U937-derived macrophages infected through antibody-dependent enhancement of dengue virus disrupts cell apical-junction complexes and increases vascular permeability. J Virol 87(13): 7486-7501.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Puerta-Guardo, H., Raya-Sandino, A., González-Mariscal, L., Rosales, V. H., Ayala-Dávila, J., Chávez-Mungía, B., Martínez-Fong, D., Medina, F., Ludert, J. E. and Angel, R. M. D. (2013). Assay to Evaluate Vascular Permeability Induction in Mice. Bio-protocol 3(22): e977. DOI: 10.21769/BioProtoc.977.
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Category
Microbiology > Microbe-host interactions > In vivo model > Mammal
Immunology > Immune cell function > Cytokine
Cell Biology > Cell staining > Whole cell
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978 | https://bio-protocol.org/en/bpdetail?id=978&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Shikimate Hydroxycinnamoyl Transferase (HCT) Activity Assays in Populus nigra
Igor Cesarino
RV Ruben Vanholme
GG Geert Goeminne
BV Bartel Vanholme
WB Wout Boerjan
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.978 Views: 13979
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in New Phytologist May 2013
Abstract
Lignin is a complex phenolic polymer deposited in secondarily-thickened plant cell walls. The polymer is mainly derived from the three primary monolignols: p-coumaryl, coniferyl and sinapyl alcohol which give rise to p-hydroxyphenyl, guaiacyl and syringyl units (H, G and S units, respectively) when coupled into the polymer. The building blocks differ in their degree of methoxylation and their biosynthetic pathway is catalyzed by more than 10 enzymes. HCT plays a crucial role by channeling the phenylpropanoids towards the production of coniferyl and sinapyl alcohols. Interestingly, HCT has been reported to be implicated in the pathway both upstream and downstream of the 3-hydroxylation of the aromatic ring of p-coumaroyl shikimate (Figure 1) (Hoffmann et al., 2003; Hoffmann et al., 2004; Vanholme et al., 2013b). These features highlight the importance of developing an assay to reliably measure HCT activity in planta. Here, we describe a UPLC-MS-based method for the analysis of HCT activity in xylem total protein extracts of Populus nigra, which can be adapted to other woody and herbaceous plant species. The protocol was initially described in Vanholme et al. (2013a).
Keywords: enzyme activity HCT Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase BAHD acyltransferase p-coumaroyl shikimate
Figure 1. The two enzymatic reactions of the phenylpropanoid pathway catalyzed by HCT. HCT (AEN02914) converts p-coumaroyl-CoA into p-coumaroyl shikimic acid (first HCT-reaction), which is converted to caffeoyl shikimic acid by C3H (coumarate 3-hydroxylase). HCT converts it further to caffeoyl-CoA (second HCT-reaction).
Materials and Reagents
Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (Biosolve, catalog number: 77-86-1 )
Dithiothreitol (DTT) (AG Scientific, catalog number: C-1029 )
Polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, catalog number: P-6755 )
Glycerol (Sigma-Aldrich, catalog number: G-7893 )
Complete Mini Protease Inhibitor Cocktail Tablets (Roche, catalog number: 0 4693159001 )
Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A-7906 )
p-coumaroyl-CoA (TransMIT, catalog number: C030 )
Shikimic acid (Sigma-Aldrich, catalog number: S-5375 )
Caffeoyl shikimate (AnalytiCon Discovery GmbH, catalog number: NP-00058 )
Coenzyme-A hydrate (CoA) (Sigma-Aldrich, catalog number: A-3164 )
Acetonitrile ULC/MS grade (Biosolve, catalog number: BIO-012041 )
Formic acid ULC/MS grade (Biosolve, catalog number: BIO-06914131 )
Liquid chromatography (LC) sample vials (Waters, catalog numbers: 186002639 and 2639531020 )
Ice
Liquid nitrogen
Protein Extraction Buffer (see Recipes)
Reaction Mix (see Recipes)
Equipment
Nunc 96-well microplate without lid and flat bottom wells (Thermo Fisher Scientific, catalog number: 269787 )
Safe-Lock tubes 2.0 ml (Eppendorf, catalog number: 3706 )
Stainless steel surgical scalpel blades No.22 (Swann-Morton, catalog number: 0 308 )
Mortar (150 x 70 mm; 700 ml) (The Morgan Advanced Materials Company plc, catalog number: 12906-6a )
Pestle (150 x 36 mm) (Haldenwanger, catalog number: 12906-2 )
Balance (Mettler Toledo, model: XP-105 Delta Range )
Vortex (IKA, model: MS2 Minishaker L002050 )
Temperature controlled benchtop microcentrifuge (Eppendorf, model: 5417R )
Temperature controlled microplate spectrophotometer (Molecular Devices, model: Spectra Max 250 )
Thermoblock (Eppendorf Thermomixer Compact) or water bath
UPLC-MS system (In our case: Waters Acquity UPLC system (WATERS) connected to a Thermo LTQ XL mass spectrometer (Thermo Fisher Scientific) or a Synapt HDMS Q-Tof (WATERS). Chromatographic separation was performed on a Waters Acquity BEH RP C18 (2.1 mm x 150 mm, 1.7 μm) column (WATERS))
Ultrafreezer
Optional: Qubit 2.0 Fluorometer (Invitrogen)
Software
XCalibur 2.0 software (Thermo Fisher Scientific, Waltham) was used to acquire, analyze and manage mass spectrometry information
Procedure
Protein extraction
Collect 15-cm stem segments derived from the base of 1-m tall sprouts emerging from the trunk of a poplar tree using secateurs. If 1-year old trees are used, segments of the main stem can be used.
Immediately submerge the fresh plant tissue into liquid nitrogen and store the samples at -80 °C until further use.
Incubate the frozen stem segments on ice for a few minutes and make a small incision in the bark on one end of the segment. The bark can be easily pealed from the underlying tissue. Some part of the cambium can stick to the debarked stem, but can be easily removed by gently scrubbing the stem with a scalpel. Scrape the xylem tissue using a scalpel, making sure the scraped tissue is immediately collected in a precooled mortar (use liquid nitrogen to keep the material frozen during sampling).
Grind the scraped xylem tissue to a fine powder using a mortar and pestle.
For xylem total protein extraction, weigh ~100 mg of ground xylem tissue in a precooled 2-ml tube and add 1 ml of ice-cold protein extraction buffer. The composition of the protein extraction buffer is given as Recipe 1, at the end of the protocol.
Vortex thoroughly and incubate on ice for 1 h, inverting the tubes every 5-10 min to prevent precipitation.
Centrifuge at 20,000 x g for 10 min at 4 °C and transfer the supernatant to a new precooled tube. Keep all samples on ice.
Protein quantification using the Bradford method (Bradford, 1976)
Prepare the BSA standards for the calibration curve as follows:
Volume of BSA (100 μg/ml)
Volume of Water
BSA Concentration
0 μl
240 μl
0 μg/ml
6 μl
234 μl
2.5 μg/ml
12 μl
228 μl
5 μg/ml
18 μl
222 μl
7.5 μg/ml
24 μl
216 μl
10 μg/ml
27 μl
213 μl
11.25 μg/ml
30 μl
210 μl
12.5 μg/ml
For the calibration curve, add 240 μl of each standard (above) to a separate well of a 96-well flat bottom microplate. All measurements are performed in triplicate.
For the quantification of xylem total protein samples, add 210 μl of water in each well and 30 μl of the protein extract dilutions in triplicate. Prepare the dilutions using the protein extraction buffer, ranging from 5x to 100x diluted (depending on the amount of plant material used for the extraction).
Add 60 μl of Bio-Rad Protein Assay reagent to each well and incubate at room temperature for 5 min.
Read absorbance at 595 nm (A595) on a plate spectrophotometer.
For the calculation of protein concentrations, generate a standard curve by plotting the BSA concentration (X-axis) versus A595 (Y-axis). After obtaining the trend line, use its corresponding equation and the absorbance of the protein sample to resolve the unknown concentration. The correlation coefficient of the trend line should be close to 1.00 (preferentially above 0.97) and all measured absorbances of the protein samples should fall into the linear range.
Notes:
Figure 2 shows an example of protein quantification using the Bradford method.
As alternative to Bradford, Qubit (Invitrogen) can be used according manufacturer’s instructions for accurate and efficient quantification of protein concentrations.
Figure 2. Total protein quantification with Bradford assay. 1Normalized absorbance is calculate by subtracting the absorbance value of the blank from the value obtained for each sample. 2Divide this value by the volume of protein extract used in the assay to calculate the final concentration (in this case, we used 2 μl).
HCT activity assay
HCT activity is measured by the conversion of p-coumaroyl-CoA and shikimate into p-coumaroyl shikimate (Figure 1).
Prior to the preparation of enzymatic reactions, boil an aliquot of the xylem protein extract for 10 min, because the boiled protein extract will be used as negative control.
The reaction mix is prepared in 1.5-ml tubes and contains 100 mM Tris-HCl pH 7, 1 mM DTT, 100 μM p-coumaroyl-CoA, 100 μM shikimic acid and 10 μg xylem protein extract (Recipe 2).
Start the reaction by adding the corresponding volume of the protein extract. Use the same amount of protein of the boiled extract as negative control.
Incubate at 30 °C for 30 min.
Note: For an in-depth analysis, different time-points can be used.
Terminate the reaction by boiling the samples for 5 min.
Note: After boiling, place the reaction tubes on ice for at least 5 min, followed by a fast spin. This brings the droplets at the lid, caused by evaporation and condensation, back into the main sample, avoiding a change in the final product concentration.
Transfer the total reaction volume (40 μl) to a LC sample vial for analysis of reaction products.
Product identification and quantification
10 μl of the aqueous phase is subjected to reversed phase LC-MS and LC-MS (Hoffmann et al., 2003). Here, the specific conditions can differ depending on the equipment used. We present our in house conditions optimized to identify and quantify p-coumaroyl shikimate on a Waters Acquity UPLC system connected to a Thermo LTQ XL mass spectrometer. Chromatographic separation is performed on an Acquity BEH C18 column (2.1 mm x 150 mm, 1.7 μm) using a gradient elution.
The mobile phase is composed of water containing 1% acetonitrile and 0.1% formic acid (Buffer A) and acetonitrile containing 1% water and 0.1% formic acid (Buffer B). The column temperature is maintained at 40 °C and the autosampler temperature at 10 °C.
A flow rate of 350 μl/min is applied during the gradient elution initializing at time 0 min 5% (B), time 30 min 50% (B), time 33 min 100% (B).
The eluent is subsequently directed to the mass spectrometer, via electrospray ionization (ESI) in negative mode.
MS source parameters are as follows: capillary temperature 300 °C, capillary voltage –24 V, source voltage 3.5 V, source current 100 μA, sheath gas flow 30, aux gas flow 20, sweep gas flow 5. The mass range is set between m/z 95-500.
The reaction product, p-coumaroyl shikimate, is characterized based on m/z 319, retention times 8.13 min (trans isomer) 9.64 min (cis isomer), and fragmentation spectra (Figure 3). Compound concentrations were quantified based on curve fitting and peak area integration of the extracted ion chromatograms for m/z 319 using XCalibur's QuanBrowser.
Figure 3. MS2 fragmentation spectra of p-coumaroyl shikimate (m/z 319)
Note: For accurate mass measurements of p-coumaroyl shikimate, a Synapt HDMS Q-Tof mass spectrometer can be used (Vanholme et al., 2013b).
Recipes
Protein Extraction Buffer
Prepare the protein extraction buffer as follows:
Stock Solution
Volume/Amount
Final Concentration
100 mM Tris-HCl pH 7.5
2 ml
20 mM
100 mM DTT
1 ml
10 mM
100% Glycerol
1.5 ml
15%
PVPP
100 mg
1%
7x Complete Mini Protease Inhibitor
1.33 ml
1x
WaterTo
10 ml
Mix thoroughly.
Notes:
Dissolve one tablet of Complete Mini Protease Inhibitor in 1.5 ml water to prepare the 7x stock solution.
Since PVPP is insoluble, it may be necessary to cut the end of the pipet tip to add the protein extraction buffer to the ground xylem material.
Reaction Mix
Prepare the enzymatic reaction mix as follows:
Stock Solution
Volume
Final Concentration
500 mM Tris-HCl pH 7.0
8 μl
100 mM
20 mM DTT
2 μl
1 mM
2 mM p-coumaroyl-CoA
2 μl
100 μM
2 mM shikimic acid
2 μl
100 μM
Xylem protein extract
x μl
10 μg
WaterTo
40 μl
Acknowledgments
The protocol was briefly described by Vanholme and coworkers in Vanholme et al. (2013a). We gratefully acknowledge funding through the European Commission’s Directorate-General for Research within the 7th Framework Program (FP7/2007-2013) under the grant agreement N° 211917 (ENERGYPOPLAR), N° 211868 (NOVELTREE) and N° 311804 (MULTIBIOPRO), the Hercules program of Ghent University for the Synapt Q-Tof (grant no. AUGE/014) and the Multidisciplinary Research Partnership ‘Biotechnology for a Sustainable Economy’ (01MRB510W) of Ghent University. RV is indebted to the Research Foundation-Flanders for a postdoctoral fellowship.
References
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Hoffmann, L., Maury, S., Martz, F., Geoffroy, P. and Legrand, M. (2003). Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism. J Biol Chem 278(1): 95-103.
Hoffmann, L., Besseau, S., Geoffroy, P., Ritzenthaler, C., Meyer, D., Lapierre, C., Pollet, B. and Legrand, M. (2004). Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis. Plant Cell 16(6): 1446-1465.
Vanholme, B., Cesarino, I., Goeminne, G., Kim, H., Marroni, F., Van Acker, R., Vanholme, R., Morreel, K., Ivens, B., Pinosio, S., Morgante, M., Ralph, J., Bastien, C. and Boerjan, W. (2013a). Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study. New Phytol 198(3): 765-776.
Vanholme, R., Cesarino, I., Rataj, K., Xiao, Y., Sundin, L., Goeminne, G., Kim, H., Cross, J., Morreel, K., Araujo, P., Welsh, L., Haustraete, J., McClellan, C., Vanholme, B., Ralph, J., Simpson, G.G., Halpin, C. and Boerjan, W. (2013b). Caffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway. Science DOI: 10.1126/science.1241602.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Cesarino, I., Vanholme, R., Goeminne, G., Vanholme, B. and Boerjan, W. (2013). Shikimate Hydroxycinnamoyl Transferase (HCT) Activity Assays in Populus nigra. Bio-protocol 3(22): e978. DOI: 10.21769/BioProtoc.978.
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Category
Plant Science > Plant biochemistry > Protein > Activity
Biochemistry > Protein > Activity
Biochemistry > Protein > Isolation and purification
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979 | https://bio-protocol.org/en/bpdetail?id=979&type=0 | # Bio-Protocol Content
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Bimolecular Fluorescence Complementation (BIFC) Protocol for Rice Protoplast Transformation
KW Kun Wang
YL Ying Liu
Shaoqing Li
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.979 Views: 15590
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in New Phytologist Apr 2013
Abstract
After the plant cells are removed the cell walls by digestive enzyme, the plant protoplasts still have good cell activity. The protoplasts can be used to transiently express proteins of target genes in living plant cells through polyethylene glycol (PEG) mediated transformation. The purpose of this method is to employ the rice protoplasts and Green fluorescent protein (GFP) as an experimental system to observe the protein interactions in vivo. Meanwhile a 505~530 nm emission filter is used in confocal microscope to eliminate the interference of the autofluorescence from plant cells. The phenomenon of plant cell body spontaneous fluorescence can be eliminated by confocal observation.
Keywords: Bimolecular Fluorescence Complementation Rice Protoplast
Materials and Reagents
Rice (Oryza sativa L.) grain seeds
Mannitol (Sigma-Aldrich)
Morpholinoethane sulfonic acid (MES) (Sigma-Aldrich)
Cellulose R-10 (Yakult Honsha)
Macerozyme R-10 (Yakult Honsha)
Bovine serum albumin (BSA) (Sigma-Aldrich)
Carbenicillin/Ampicillin (AMRESCO)
β-Mercaptoethanol (β-ME) (Amresco)
Polyethtlene glycol (PEG) 4000 (Sigma-Aldrich)
Enzyme solution (see Recipes)
PEG4000 solution (see Recipes)
W5 solution (see Recipes)
MMG solution (see Recipes)
Equipment
Shaker P270 (Chinese Academy of Sciences, Wuhan Scientific Instrument Plant)
Collagen-coated 35-mm-diameter glass-base dish
Vortex XW-80A (JiaPeng Techno)
Nylon mesh (35 μm) (EMD Millipore)
Vacuum pump
50 ml with round bottom centrifuge tube
2 ml centrifuge tube
Tabletop centrifuges (Eppendorf 5810R and 5417R )
Collagen-coated 35-mm-diameter glass-base dish (Asahi Techno Glass Corporation)
Confocal microscopy (Olympus, model: FV1000 )
OLYMPUS FV1000 system (Fluoview Ver.1.7b Viewer) (Olympus)
Procedure
Protoplast Preparation
Germinate 100 of the sterilizing rice grain seeds on wet gauze under darkness at 28 °C for about one week.
When the etiolated seedlings grow to about 7~8 cm, collect the etiolated seedlings, and immediately use a sharp blade to cut the seedlings into ~0.5 mm segments, then, have the segments fully immersed in 50 ml 0.6 M mannitol solution for 10 min.
Transfer the seedling fragments into Enzyme Solution.
Using vacuum pump to remove air in the tissues to help them being completely precipitated in the Enzyme Solution.
Keep the tissue-immersed solution in darkness at 28 °C, and agitated at 80 rpm on a shaker for ~4 h.
Wash the nylon net (35 μm) with ddH2O and then wet it with W5 solution for 3-5 min before filtering the protoplast, and then the enzyme digested samples are filtered to a 50 ml centrifuge tube with round bottom. Slightly twist the nylon net to improve the yield.
Centrifuge the filtration at 100 x g for 5 min, discard the supernatant and remove the residual liquid as much as possible with pipette, then add 10 ml pre-cooled W5 solution to resuspend the protoplast pellet by gentle swirling.
Incubate the tube for 30 min on ice (the following operations are under room temperature).
Precipitate the protoplast by centrifugation (100 x g for 5 min). Discard the supernatant and remove the residual liquid, then gently add 1 ml MMG solution to resuspend the protoplasts. Finally, adjust the protoplast density to 2 x 105 cells/ml under microscope (40x).
Protoplast Transformation
Note: Before protoplast transformation, please prepare the BIFC expression vectors according to the protocols of Walter et al. (2004), and we recommend to refer the information on how to prepare plasmid DNA using the economical CsCl gradient on the website of Sheen lab (http://genetics.mgh.harvard.edu/sheenweb/protocols.html).
Aliquot 100 μl of the protoplasts to 2 ml centrifuge tubes.
For transformation, empty vectors pUC-SPYNE/pUC-SPYCE and bZIP6-YFPN/bZIP6-YFPC are used as negtive and positive controls, respectively. 20 μl of the BIFC vectors (≥ 1-2 μg/μl, 10 μl per vector), negative control and positive control are added to each tube, respectively, and then mix gently.
Add equal volume (120 μl) PEG solution to each tube and mix well.
Incubate the mixture for 15 min for transformation.
Add 480 μl of W5 solution to stop the transformation.
Centrifuge the solution at 100 x g for 2 min, and discard the supernatant.
Add 1 ml W5 solution to gently resuspend the protoplast pellet, and add 1 μl Carbenicillin (50 mg/ml) before transferring the protoplasts to culture plate, culture at room temperature for 16-20 h in darkness to allow expression of the BIFC proteins.
Before confocal observation, the transformed protoplasts should be centrifuged at 100 x g for 2 min and remove most of the supernatant, then resuspend the protoplasts.
Confocal observation
Transfer the protoplast into a collagen-coated 35-mm-diameter glass-base dish for microscopy observation.
Collection of the confocal fluorescence signals was performed on Olympus FV1000 system.
The interference from autofluorescence problem in experiment can be eliminated by optical sectioning generated in confocal microscopy. We choose using excitation with the 488-nm line of an argon laser and a 505~530 nm band-pass emission filter.
Under this observation regime, the positive control show strong yellow fluorescence, and the negative control is black. This confirms all of the operations above are reliable for the BIFC observation.
Recipes
Enzyme solution (10 ml)
Mannitol (0.6 M)
1.093 g
MES (10 mM, pH 5.7)
1 ml (100 mM stock solution)
Cellulose R-10 (1.5%)
0.15 g
Macerozyme R-10 (0.75%)
0.075 g
BSA (0.1%)
0.01 g
CaCl2 (1 mM)
0.1 ml (100 mM stock solution)
Carbenicilli (0.25 g/ml)
2 μl
β-ME
4 μl
Add ddH2O to 10 ml
55 °C 10 min
Natural cooling (Preparing it when you use)
PEG4000 solution
Mannitol (0.6 M)
1.093 g
CaCl2 (100 mM)
0.111 g
PEG4000 (40%)
4 g
Add ddH2O to 10 ml
Using 1 M KOH to adjust the pH to 7.5~8.0
Aliquot with 1.5 ml centrifuge tube and preserve at -20 °C
W5 solution
W5 (100 ml)
154 mM NaCl
NaCl
0.9 g
125 mM CaCl2
CaCl2
1.39 g
5 mM KCl
KCl
5 ml 100 mM stock solution
5 mM glucose
Glucose
0.09 g
2 mM MES
MES
2 ml 100 mM stock solution
Adjust pH to 5.8 with KOH, High temperature and high pressure sterilization for 20 min, room temperature preservation
MMG solution
MMG solution (10 ml)
15 mM MgCl2
MgCl2
1.5 ml 100 mM stock solution
4 mM MES
MES
0.4 ml 100 mM stock solution
0.6 M Mannitol
Mannitol
1.093 g
Adjust pH to 5.8 with KOH, High temperature and high pressure sterilization for 20 min, room temperature preservation
Acknowledgments
This protocol is adapted from Wymer et al. (1999); Walter et al. (2004); Yoo et al. (2007) and Whang (2009).
References
Wymer, C. L., Beven, A. F., Boudonck, K. and Lloyd, C. W. (1999). Confocal microscopy of plant cells. Methods Mol Biol 122: 103-130.
Whang, S. S. (2009). Confocal microscopy study of Arabidopsis embryogenesis using GFP: mTn. J Plant Biol 52(4): 312-318.
Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., Harter, K. and Kudla, J. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40(3): 428-438.
Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Wang, K., Liu, Y. and Li, S. (2013). Bimolecular Fluorescence Complementation (BIFC) Protocol for Rice Protoplast Transformation. Bio-protocol 3(22): e979. DOI: 10.21769/BioProtoc.979.
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Category
Plant Science > Plant cell biology > Cell imaging
Biochemistry > Protein > Interaction > Protein-protein interaction
Cell Biology > Cell imaging > Fluorescence
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98 | https://bio-protocol.org/en/bpdetail?id=98&type=1 | # Bio-Protocol Content
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Peer-reviewed
Cell Adhesion Assay
YC Yanling Chen
Published: Mar 5, 2012
DOI: 10.21769/BioProtoc.98 Views: 70198
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Abstract
Cell adhesion, the binding of a cell to the extracellular matrix (ECM), other cells, or a specific surface, is essential for the growth and survival of the cell and also its communication with other cells. The process of cell adhesion involves a range of biological events such as three-dimensional re-organization of the cytoskeleton, biochemical reactions in the cell, and changes in molecules on the surface of the cell. Cancer cells, especially the highly metastatic types, are believed to have enhanced adhesion ability that often facilitates the migration of the cells to a new site to establish new tumors in the body. Cell adhesion assay is therefore often used to evaluate the metastatic ability of cancer cells. In addition, the assay can also be used to assess the effect of certain treatment (e.g., exposure to chemicals) on the ability of cells to adhere. A modified cell adhesion assay protocol is described here for studying the interactions between cells and extracellular materials.
Materials and Reagents
Hela cells (ATCC, catalog number: CCL-2 ™)
MTT cell proliferation assay kit (ATCC, catalog number: 30-1010K ™)
Collagen I (Sigma-Aldrich, catalog number: C7661 )
Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
0.5 M EDTA solution (pH 8.0) (Life Technologies, Invitrogen™/Ambion®, catalog number: AM9260G )
Bovine serum albumin (BSA) (Life Technologies, Invitrogen™, catalog number: 15561-020 )
Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
Equipment
Corning 96-well polystyrene plate (Fisher Scientific, catalog number: 07-200-91 ; Corning Incorporated, catalog number: 3598 )
Cell culture incubator: 37 °C and 5% CO2
Spectrophotometer that can measure absorption at 570 nm with 96-well format
Procedure
Grow the Hela cells in DMEM supplemented with 10% FBS.
Prepare 40 μg/ml Collagen I solution in PBS, store at 4 °C; prepare 0.1% BSA solution in DMEM.
Coat the 96-well plate (30 μl/well) with the Collagen I solution at 4 °C.
After 12 h of coating, remove the Collagen I solution and air-dry the plate at room temperature in the tissue-culture hood.
Deprive cells of serum for 8 h before the adhesion assay. To do so, wash cells three times with serum-free DMEM and grow them in DMEM.
Use 10 mM EDTA in DMEM to detach the cells and then observe them under a microscope to confirm complete dissociation of the cells, which would take ~10 min.
Wash cells twice with DMEM to remove EDTA, resuspend cells at 2 x 105 cells/ml in DMEM with 0.1% BSA.
For cell-substratum adhesion assay, add 100 μl cell suspension (from step 7) to each of the Collagen I-coated wells. Incubate the plate at 37 °C for 20 min to allow the cells to adhere to the surface.
Add 100 μl DMEM to each well to wash off any non-adherent cells, wash four times.
Note: To achieve consistency, always add/remove DMEM gently with multi-channel pipetter for multiple wells.
After washing, add DMEM with 10% FBS and incubate the cells at 37 °C for 4 h for recovery.
Add 10 μl of MTT substrate to each well and continue incubation for an additional 2 h at 30 °C.
Next, lyse the MTT-treated cells in 100 µl DMSO (or other lysis buffer of choice) and measure absorbance at 570 nm on a spectrophotometer (see Note 1).
Notes
Consider including the following reference group for monitoring each step of the procedure: Wells not coated with Collagen I; wells not washed with DMEM; wells not added with cells; wells not added with MTT (background for MTT assay).
Acknowledgments
This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Chen et al. (2009), Humphries et al. (1998) and Mobley and Shimizu (2001). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee [see Chen et al. (2009)].
References
Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
Humphries, M. (1998). Curr Protoc Cell Biol 9.1.1-9.1-11.
Mobley, J. and Shimizu, Y. (2001). Curr Protoc Immunol Chapter 7: Unit 7.28.
Article Information
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© 2012 The Authors; exclusive licensee Bio-protocol LLC.
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Category
Cancer Biology > General technique > Cell biology assays
Cancer Biology > Invasion & metastasis > Drug discovery and analysis
Cell Biology > Cell structure > Cell adhesion
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980 | https://bio-protocol.org/en/bpdetail?id=980&type=0 | # Bio-Protocol Content
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Peer-reviewed
Binding Assay of Cytosolic Proteins to the Cytoskeleton
Stefano Del Duca
Giampiero Cai
Published: Vol 3, Iss 22, Nov 20, 2013
DOI: 10.21769/BioProtoc.980 Views: 12024
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Plant Physiology Apr 2013
Abstract
Many cellular proteins interact with the cytoskeleton (both actin filaments and microtubules), either dynamically or permanently. This interaction is required during different aspects of the cell life, for example during the process of cell division. In addition, many enzymes interact transiently with actin filaments and microtubules in order to promote their cellular distribution. Several substances with inhibitory capacity can affect this binding and cause damages to cells. This protocol allows to analyze whether a protein interacts with either actin filaments or microtubules and, when applicable, the conditions controlling this interaction.
The test is based on the specific binding between the protein of interest and the cytoskeletal filaments. As shown schematically in the diagram of example (see below), the test starts from the cell lysate to which actin filaments (produced from monomeric actin) are added. The mixture (performed under different experimental conditions chosen by the operator) is then incubated so that the protein of interest (in the example, myosin) binds to actin filaments. The sample is then centrifuged in order to separate unbound or weakly-bound proteins from actin filaments to which both the protein of interest and, eventually, traces of less specific proteins are associated.
Keywords: Actin filament Microtubule Myosin Protein binding
Materials and Reagents
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (≥ 97.0%) (Sigma-Aldrich, catalog number: E3889 )
Guanosine 5’-triphosphate sodium salt hydrate (GTP) (≥ 95%) (Sigma-Aldrich, catalog number: G8877 )
DL-Dithiothreitol (DTT) (≥ 99.0%) (Sigma-Aldrich, catalog number: 43819 )
Adenosine 5’-triphosphate magnesium salt (ATP) (95-98%) (Sigma-Aldrich, catalog number: A0770 )
PIPES disodium salt (≥ 99%) (Sigma-Aldrich, catalog number: P3768 )
Glycerol (≥ 99%) (Sigma-Aldrich, catalog number: G5516 )
MgCl2 (≥ 98%) (anhydrous) (Sigma-Aldrich, catalog number: M8266 )
Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S9378 )
Taxol (Paclitaxel from Taxus brevifolia) (≥ 95%) (Sigma-Aldrich, catalog number: T7402 )
Bovine Tubulin (lyophilized, > 99% pure) (CYTOSKELETON, catalog number: TL238-B )
Actin protein from rabbit skeletal muscle (lyophilized, > 99% pure) (CYTOSKELETON, catalog number: AKL99 )
2D Quant Kit (General Electric Company) or Bradford Protein Assay kit (Bio-Rad Laboratories)
Tubulin Dilution Buffer (TDB) (see Recipes)
Glycerol buffer (GB) (see Recipes)
“Sucrose cushion” buffer (SC) (see Recipes)
General actin buffer (A-buffer) (see Recipes)
Polymerization Inducer (PI) (see Recipes)
Cushion buffer for F-actin (see Recipes)
Equipment
Apparatus for the lysis of cells and tissues (the type of apparatus for cell lysis depends on the cellular system with which one is working; animal cells can be lysed easily while plant cells, because of the presence of the cell wall, require more energetic methods such as freezing under liquid nitrogen and powdering with pestle and mortar. It is therefore not possible to give precise indications but we prefer to leave the choice to individual operators.)
Electrophoretic apparatus (Mini-PROTEAN® II Electrophoresis Cell) (Bio-Rad Laboratories)
Ultracentrifuge (Beckman Coulter, model: Optima LE-80 K) equipped with a 70 Ti fixed-angle rotor and adapters for 4-ml polyallomer tubes
Bio-Rad Mini-Trans-Blot Cell Apparatus (optional)
Software
ImageJ or Bio-Rad Quantity One
Procedure
Microtubule binding assay
Preparation of the soluble (cytoplasmic) extract
This step necessarily depends on the cells or tissues on which one is working. In theory, soft tissues (such as animal tissues) can be lysed directly in the lysis buffer using either plastic Eppendorf pestles or an Ultra-Turrax homogenizer in case of complex tissues. For plant cells, it is preferable to freeze the tissue of interest under liquid nitrogen and then grind it with pestle and mortar. After the tissue was completely powdered, the liquid nitrogen is allowed to evaporate. The tissue powder is moved to a suitable tube, like eppendorf tubes or 15-ml conical tubes (depending on the amount of starting material). The lysis buffer is added and the sample is incubated at 4 °C or on ice for 15 min with gentle agitation. At the end, the sample is centrifuged at high speed (> 100,000 x g) for pelleting cellular debris and for getting cytosolic proteins in the supernatant. Although the extract of soluble proteins can be frozen under liquid nitrogen and stored at -80 °C, we always prefer to work with fresh material because it is difficult to estimate the temporal stability of the extract. The composition of lysis buffer may be adjusted from species to species, please refer to the literature on the specific tissue in order to determine the best lysis buffer.
Determine protein concentration of the cytoplasmic extract in order to mix known protein quantities. Several commercial kits are available for determination of protein concentration (such as the 2D Quant Kit or the Bradford Protein Assay kit). Always take an aliquot for electrophoresis.
Preparation of microtubules
Thaw one aliquot of lyophilized tubulin (animal or plant source, 1 mg), then add 200 μl of TDB so that the final concentration is 5 mg/ml.
Add 25 μl of GB and incubate at 35 °C for 20 min.
Mix 1.8 ml of TDB with 81 μl of 500 μM taxol (TDB-T). Incubate at 35 °C for 15-20 min in such a way that the temperature of the taxol solution is the same of tubulin.
At the end of incubation, add 1.8 ml of TDB-T to 225 μl of the tubulin sample. Mix gently. Tubulin is now in a final volume of ≈ 2 ml at a concentration of 0.5 mg/ml.
Prepare additional TDB-T by mixing 1.8 ml of TDB with 81 μl of 500 μM taxol.
Preparing the binding mix.
Mix the cytosolic extract (in variable amounts, for example from 0 to 40 microliters, in order to work with a range of 10-100 micrograms of protein) with a constant volume of the microtubule sample (40 microliters). Use the remaining volume for the addition of other substances (i.e. inhibitors), and then adjust the volume to 100 microliters with TDB-T buffer. Volumes can be increased correspondingly to accommodate for different rotors and tubes.
Incubate the samples at room temperature for 30 min.
The test temperature can be adjusted if it is a variable examined in the experiment.
Separation of cytoskeletal filaments
Centrifuge the samples at 100,000 x g for 40 min at 20 °C over 0.5 ml of sucrose cushion buffer.
After centrifugation, the supernatant is located above the sucrose cushion and may therefore be removed with a pipette without touching the sucrose cushion. Process the supernatants for electrophoresis.
Resuspend the pellets directly into a denaturing buffer suitable for electrophoretic analysis (e.g. Laemmli buffer) (max. 100 microliters).
Analysis
Test individual fractions (supernatants and pellets) by electrophoresis (SDS-PAGE) and possibly by western blot, the latter being required to identify specifically and to quantify the protein of interest.
Actin filament binding assay
Preparation of cell lysate
It depends on the particular tissue or cells on which one is working. See the previous protocol for more details.
Determine the protein concentration of samples using commercial kits (such as the 2D Quant Kit or the Bradford Protein Assay kit) and always collect one aliquot for electrophoresis.
Preparation of actin filaments
Thaw one aliquot of lyophilized actin (animal or plant sources, 1 mg).
Dilute the actin to a concentration of 10 mg/ml by adding 100 μl of A-buffer. Remove solution from the tube (usually an Eppendorf tube) and place in 5-ml or 15-ml tubes.
Dilute the actin at a concentration of 0.4 mg/ml with A-buffer (100 μl of actin + 2.4 ml of A-buffer). Incubate on ice for 1 h (agitation is not required).
Add the Polymerization Inducer 1x final: 2.25 ml + 0.25 ml of actin Polymerization Inducer 10x. Incubate at room temperature for 1 h.
Preparing the binding mix.
Mix the cytosolic extract (in variable amounts, for example from 0 to 40 microliters, in order to work with a range of 10-100 micrograms of protein) with a constant volume of the actin filament sample (50 microliters). Use the remaining volume for the addition of other substances (i.e. inhibitors), and then adjust the volume to 100 microliters with the A-buffer/Polymerization Inducer mix (which is the same buffer of the actin solution). Volumes can be increased correspondingly to accommodate for different rotors and tubes.
Incubate samples at room temperature for 30-60 min.
The test temperature can be adjusted if it is a variable examined in the experiment.
Separation of cytoskeletal filaments
Centrifuge samples at 150,000 x g for 60-90 min at 20 °C over 0.5 ml of the cushion buffer for F-actin.
Take the supernatants and process them for electrophoresis.
Resuspend the pellets in 100 μl of a denaturing buffer suitable for electrophoretic analysis (e.g. Laemmli buffer).
Analysis
Test individual fractions (supernatants and pellets) by electrophoresis (SDS-PAGE) and possibly by western blot, the latter being required to identify specifically and to quantify the protein of interest (Figure 1).
In both cases, the relative quantification of bands after western blot analysis can be performed using image acquisition systems and specific software (free like ImageJ or paid such as the Bio-Rad Quantity One).
Figure 1. Example of binding analysis with actin filaments. Specifically, the protein myosin from rabbit skeletal muscle (arrow) was incubated with filamentous actin (arrowhead). S and P indicate the supernatants and pellets, respectively. Lane 1, molecular weight standard, the value of which is indicated by numbers on the left. Lane 2, myosin (commercially available). Lanes 3-4, supernatant and pellet obtained after incubation of myosin with actin filaments. Lanes 5-6, supernatant and pellet obtained after incubation of myosin with actin filaments in the presence of 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP, a non-hydrolyzable analogue of ATP). Lanes 7-8, supernatant and pellet obtained after incubation of myosin with actin filaments in the presence of ATP. Note that myosin binds weakly in the presence of ATP but very strongly in the presence of AMPPNP.
Recipes
Tubulin Dilution Buffer (TDB)
80 mM Pipes, pH 6.8
1 mM EGTA
1 mM MgCl2
2 mM GTP
Glycerol buffer (GB)
80 mM Pipes, pH 6.8
1 mM EGTA
1 mM MgCl2
60% (w/v) glycerol
“Sucrose cushion” buffer (SC)
80 mM Pipes, pH 6.8
1 mM EGTA
1 mM MgCl2
10% (w/v) sucrose
20 μM taxol
General actin buffer (A-buffer)
5 mM Tris-HCl pH 8.0
0.2 mM CaCl2
0.2 mM ATP
0.5 mM DTT
10x Polymerization Inducer (PI)
500 mM KCl
20 mM MgCl2
10 mM ATP
Cushion buffer for F-actin
5 mM Tris-HCI pH 8.0
2 mM MgCl2
50 mM KCl
10% (v/v) glycerol
Acknowledgments
The protocol was adapted from a previously published paper: Del Duca et al. (2013). The work was supported by PRIN 2007 (grant no. 2007RZCW5S_003) and PRIN 2008 (grant no. 2008BK7RXB), funded by the Italian Ministry of University and Research, and by Bologna University (RFO 2010 [grant no. RFO10DELDU] and RFO 2011 [grant no. RFO11DELDU]) to S.D.D.
References
Cai, G., Faleri, C., Del Casino, C., Emons, A. M. and Cresti, M. (2011). Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules. Plant Physiol 155(3): 1169-1190.
Del Duca, S., Faleri, C., Iorio, R. A., Cresti, M., Serafini-Fracassini, D. and Cai, G. (2013). Distribution of transglutaminase in pear pollen tubes in relation to cytoskeleton and membrane dynamics. Plant Physiol 161(4): 1706-1721.
Del Duca, S., Serafini-Fracassini, D., Bonner, P., Cresti, M. and Cai, G. (2009). Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments. Biochem J 418(3): 651-664.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Del Duca, S. and Cai, G. (2013). Binding Assay of Cytosolic Proteins to the Cytoskeleton. Bio-protocol 3(22): e980. DOI: 10.21769/BioProtoc.980.
Del Duca, S., Faleri, C., Iorio, R. A., Cresti, M., Serafini-Fracassini, D. and Cai, G. (2013). Distribution of transglutaminase in pear pollen tubes in relation to cytoskeleton and membrane dynamics. Plant Physiol 161(4): 1706-1721.
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Category
Plant Science > Plant cell biology > Cell structure
Biochemistry > Protein > Interaction > Protein-protein interaction
Plant Science > Plant biochemistry > Protein > Interaction
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981 | https://bio-protocol.org/en/bpdetail?id=981&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol
Elizabeth V. Clarke
MB Marie E. Benoit
AT Andrea J. Tenner
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.981 Views: 11269
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Original Research Article:
The authors used this protocol in The Journal of Immunology Jun 1986
Abstract
Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described previously (Bobak et al., 1986). From a unit of blood drawn into anticoagulant, 60-120 million monocytes can be obtained. These cells are not activated and have been shown to be appropriately capable of differential activation in multiple studies.
Keywords: Monocyte Human Isolation Elutriation Lymphocyte
Materials and Reagents
Peripheral blood from normal human donor (450 ml)
CPD AS-1 blood collection bags (Citrate-Phosphate-Dextrose, Adenine saline-1) (Baxter)
1x PBS-2mM EDTA
Sterile milliQ H2O
Lymphocyte separating medium (LSM) (MP Biomedicals, catalog number: 0 850494 )
HBSS (Fisher Scientific, catalog number: 21023CV )
Ethanol (70%, 1 liter)
10% Bleach (1 liter)
10% defined FBS (Hyclone, catalog number: SH30070.03 )
1% P/S (Mediatech, CellGro®, catalog number: 30-002-CIRF )
1% L-Gln (Mediatech, CellGro®, catalog number: 25-005-CI )
Recombinant human IL-2 (Pepro Tech, catalog number: 200-02 )
Recombinant human M-CSF (Pepro Tech, catalog number: 300-25 )
25% Albumin (Human), USP (Talecris Plasma Resources, catalog number: 13533-684-20 )
Complete media (see Recipes)
1x sterile PBS (dilute 1:5 from 5x PBS pH 7.0 – 7.2) (see Recipes)
Ammonium Chloride Potassium (ACK) buffer (see Recipes)
1x sterile Elutriation Buffer (see Recipes)
5x Phosphate Buffered Saline (see Recipes)
Equipment
Extruder
T75 flasks
Nunc blue cap 50-ml polypropylene tubes (important as monocytes stick to other tubes lowing yields)
Tubing clamps
Scissors
P1000 Pipetteman
Petri dishes
250 ml plasma tube
Isopropanol wipes, individually packaged
Beckman JE6B Elutriation System and Rotor (Beckman Coulter, model: JE6B-IM-3) (November 1992)**
Centrifuge (Sorvall, model: RT6000 or RC-3B )
Stericup (Fisher Scientific, catalog number: SCGVU11RE )
** Beckman Coulter has newer models Avanti J-301 or J-26S XP with Elutriation Systems, but we have no experience with those.
Procedure
Receive 450 ml peripheral blood from normal human donor drawn in CPD AS-1
Using an RC-3B Centrifuge, spin a 450 ml bag of blood upright with no brake, 25 °C, 1,665 x g, for 7 min. Takes approximately 30 min to come to a stop without the brake.
Assemble elutriator and chambers
See Beckman JE6B Elutriation System and Rotor Assembly Manual.
Sterilize and prepare elutriation chamber
Attach 1 ml pipet to elutriation pump tubing and transfer tubing (by submerging in each solution) to each of the following in sequence:
Wash #1 10% fresh bleach (in sterile milliQ H2O)
Wash #2 300-500 ml: sterile milliQ H2O
Wash #3 300-500 ml: 70% EtOH (sterile milliQ H2O)
Spin elutriator rotor by hand for 30 sec to remove air pockets.
Wash #4 300-500 ml: sterile milliQ H2O
Wash #5 300-500 ml: sterile PBS
Finally, equilibrate tubing and chamber with elutriation buffer (with 100-200 ml approximately 11-12 ml/min.flow rate).
Collect blood from centrifuge
Sit blood bag on extruder carefully and place onto the hooks on extruder.
Wipe tube of blood bag and scissors with alcohol wipes to sterilize. Wipe tubing end with alcohol, attach clamp, and insert into a 250 ml plasma tube (if collecting plasma for later use) or waste (if not collecting plasma).
With clamp shut on tubing, release press to squeeze bag gently.
Open clamp to control flow to a fast drip/slow stream. Stop plasma collection when plasma is approximately 1 inch from top of bag.
Transfer blood bag tube to T75 flask containing 50 ml PBS 0.02 mM EDTA, where you will collect approximately 25 ml plasma and 20 ml erythrocytes plus white cells) often called the buffy coat. Swirl flask gently. Total volume in flask should be approximately 90-100 ml.
Tilt 50 ml tube containing 14-18 ml LSM and using a 10 ml pipette, layer approximately 30 ml buffy coat-PBS-EDTA carefully from flask onto LSM, repeat with second and third LSM tube.
Remainder of buffy coat-PBS-EDTA should be layered onto last LSM tube.
Balance LSM tubes, spin in RT6000 at 350 x g for 40 min, 22 °C with brake off.
Blood/cells
Remove diluted Plasma from top of tubes of LSM (approximately 25 ml).
Figure 1. Adapted from http://www.genec.cl/stock/Mediatech.html
Remove peripheral blood mononuclear cells (PMBC) interfaces, add to 40 ml PBS/1 mM EDTA in 50 ml tubes and invert to mix.
Spin in RT6000 to remove platelets, 200 x g for 10 min. Pellet should be visible.
Pour off most of the supernatant.
Resuspend all pellets, pooling in a total of 8–10 ml elutriation buffer. Avoid air bubbles.
Count PBMCs: should be between 7 x 108 and 1.5 x 109 total cells depending on the donor.
Elutriation
Run some elutriation buffer through to equilibrate (100 ml).
Spin rotor manually for approximately 30 sec to remove air pockets.
Close lid and allow speed to get up to 1,450 x g (final speed) before checking flow rate: should be between 11.4 and 11.6 ml/min.
Transfer pipette attached to pump tubing into cells and pump full volume of cells (8-10 ml) before transferring pipette back to elutriation buffer. Avoid taking up any air by pinching tubing during transfer.
Collect the effluent. Collect the first two 50 ml tubes of effluent which will contain the lymphocyte population (with some contaminating platelets and red cells) and spin down at 100 x g to remove platelets. Lyse any remaining erythrocytes in ACK at room temperature (by resuspending the pellet in the ACK and gently inverting the tube 1-2 times), wash the lymphocytes once in HBSS by resuspending in 25 ml, count cell concentration, pellet cells again at 100 x g, and resuspend the lymphocytes in complete media containing 50 U/ml IL-2 at 2 million/ml in complete media in T75 flasks. Monocytes will remain in chamber, due to size and density and centrifugation conditions.
After 35 min run at 11.5 ml/min increase speed to 12.5 ml per min. Continue to run for 25 min (total runtime is 60 min).
End of run
Clamp output and input tubes before pump is turned off, then immediately stop centrifuge.
Open lid and unlock black lock, then remove input tube first, then lower outlet tube.
Remove elute chamber, with spout up.
In hood, using a P1000 remove the 6.3 ml of sample from the elutriation chamber and add to 50 ml tube.
Bring up volume up to 20 ml with 1x PBS. Count cells. Monocytes should be approximately 10-20% of original PBMC count. Wash cell pellet with sterile PBS. 200 x g for 10 min. Resuspend pellet to 100 x 106 cells/ml.
Plate monocytes in 10 cm petri dishes at 0.5 x 10 E6/ml (10 ml total/dish) in complete media containing 25 ng/ml M-CSF for derivation into macrophages.
Clean elutriator
Place elute chamber back into rotor and rotor back into centrifuge chamber attaching tubes to wash out/decontaminate rotor, tubing and chamber.
Undo clamps and run through 300 ml 10% bleach, 300 ml autoclaved H2O, 300 ml 70% EtOH, 300 ml water again. Disassemble rotor, set in 70% ethanol for 30% and let air dry overnight.
Recipes
Complete media
RPMI
10% defined FBS
1% P/S
1% L-Gln
ACK buffer
To 450 ml milliQ water, add :
4.145 g Ammonium Chloride (annhydrous)
0.5 g potassium bicarbonate
18.6 mg disodium EDTA
Adjust pH to 7.4
Bring final volume to 500 ml with milliQ water
Fliter sterilize (Using Stericup) and store at 4 °C.
1x sterile Elutriation Buffer, pH 7.3
8.3 g NaCl
5 g dextrose in 1 L MQ H2O
10 mM Na2HPO4
3 mM NaH2PO4.H2O
100,000 units Pen/Strep
0.625% Human Serum Albumin (HSA) from Talecris Therapeutics
5x Phosphate Buffered Saline (PBS for washing cells), pH 7.0-7.2
9.51 g dibasic phosphate, anhydrous (diluted is 6.7 mM Na2HPO4)
4.56 g monobasic phosphate (diluted is 3.3 mM Na H2PO4-H2O)
85 g NaCl (diluted will be .14 M)
Dissolve and bring to final volume of 2000 ml with MilliQ (LPS-free) water. Filter sterilize. Diluted at room temperature should be pH 7.0-7.2, and K = 14-16. Store at 4 °C.
Acknowledgments
The original protocol was published in Bobak et al. (1986). Further modification and development of these methods was supported in part by NIH AI-41090 and T32 AI 60573.
References
Bobak, D. A., Frank, M. M. and Tenner, A. J. (1986). Characterization of C1q receptor expression on human phagocytic cells: effects of PDBu and fMLP. J Immunol 136(12): 4604-4610.
Lionetti, F., Hunt, S. and Valeri, C. (1980). Methods of cell separation. Plenum Publishing Corporation, New York.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Clarke, E. V., Benoit, M. E. and Tenner, A. J. (2013). Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol. Bio-protocol 3(23): e981. DOI: 10.21769/BioProtoc.981.
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Category
Immunology > Immune cell isolation > Lymphocyte
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982 | https://bio-protocol.org/en/bpdetail?id=982&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Adherent-invasive Escherichia coli Biofilm Formation Assays
BC Benoit Chassaing
AD Arlette Darfeuille-Michaud
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.982 Views: 13136
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Journal of Bacteriology Jan 2013
Abstract
Patients with Crohn’s disease are abnormally colonized by adherent-invasive Escherichia coli (AIEC) bacteria (Chassaing and Darfeuille-Michaud, 2011). These bacteria are able to adhere to and invade intestinal epithelial cells (IEC), to replicate within macrophages, and were recently described to be able to form biofilms (Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013). The reference strain of adherent-invasive E. coli is the strain LF82, associated with ileal Crohn’s disease (Darfeuille-Michaud et al., 1998).
This protocol described basic steps of a biofilm formation assay on I) non-cell-treated polystyrene microtiter plates and on II) paraformaldehyde-fixed I-407 IEC monolayers.
Keywords: Inflammatory Bowel Disease Adherent-Invasive Escherichia coli Biofilm
Materials and Reagents
Adherent-invasive E. coli reference strain LF82 (Darfeuille-Michaud et al., 1998)
Intestine-407 (I-407) cells (ATCC, catalog number: CCL-6 )
Luria Broth (BD DifcoTM, catalog number: 244620 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
M63 medium (United States Biological, catalog number: M1015 )
MgSO4 (Sigma-Aldrich, catalog number: 208094 )
Phosphate-Buffered Saline (PBS) ( Mediatech, Cellgro®, catalog number: 21-040 )
Crystal Violet (Sigma-Aldrich, catalog number: C6158 )
200 proof absolute Ethanol (Sigma-Aldrich)
Fetal bovine serum (FBS) (Mediatech, Cellgro®, catalog number: 35-010-CV )
Nonessential amino acids (Mediatech, Cellgro®, catalog number: 25-025-CI )
L-glutamine (Mediatech, Cellgro®, catalog number: 25-005 )
Penicillin/Streptomycin/Amphotericin B solution (Mediatech, Cellgro®, catalog number: 30-004-CI )
Vitamin mix (Mediatech, Cellgro®, catalog number: 25-020-CI )
Formaldehyde solution (Sigma-Aldrich, catalog number: F8775 )
Phalloidin-tetramethyl rhodamine isocyanate (Sigma-Aldrich, catalog number: P1951 )
Paraformaldehyde
Hoechst 33258 (Sigma-Aldrich, catalog number: B1155 )
Vectashield (Vector Labs, catalog number: H-1000 )
Minimum Essential Medium (MEM) (Mediatech, Cellgro®, catalog number: 10-022-CV ) (see Recipes)
Equipment
Coverslips (Electron Microscopy Sciences)
Sterile tubes
24-well tissue culture plates
Non-cell-treated polystyrene 96-well microtiter plates (Falcon®, catalog number: 62406-117 )
24-well polystyrene plate, tissue-culture treated (Falcon®, catalog number: 62406-159 )
Microplate shaker (LABREPCO, model: BT1500 )
Microplate spectrophotometer
Confocal microscope
30 °C incubator
37 °C/5% CO2 incubator
Software
Computer program COMSTAT1
Procedure
Biofilm formation assay on non-cell-treated polystyrene microtiter plates
Bacterial strains were grown overnight in sterile tubes containing 2 ml of Luria-Bertani broth with 5 g.L-1 glucose at 35.5 °C without agitation (Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013).
The following day, bacterial suspension was diluted 1/100 in M63 minimal medium supplemented with 1 mM of MgSO4 and 8 g.L-1 glucose.
Aliquots of 130 μl were then placed in wells of non-cell-treated polystyrene 96-well microtiter plates and incubated 24 h at 30 °C without shaking. One well designed as blank received only M63 minimal medium supplemented with 1 mM of MgSO4 and 8 g.L-1 glucose without bacteria.
After incubation, microplates were agitated for 5 min using a microplate shaker, and bacterial growths were then estimated by OD620nm reading using a microplate spectrophotometer.
The liquid was remove by decantation, and the wells were washed once using 200 μl sterile PBS without agitation.
In order to dry them, plates were left open for at least 20 min at room temperature.
Adherent bacteria forming biofilm were stained with 130 μl of 1% crystal violet solubilized in ethanol for 5 min at room temperature without agitation.
Wells were then washed five times using 200 μl sterile PBS without agitation.
In order to dry them, plates were left open for at least 1 h at room temperature.
130 μl of absolute ethanol was added, and plates were incubated 5 min at room temperature without agitation.
High-speed agitation was performed using a microplate shaker for 10 min (1000 rpm).
Biofilm formation was estimated by OD570nm reading using a microplate spectrophotometer.
13.Specific biofilm formation index could be determined by using the ration OD570nm/OD620nm that allows standardization of biofilm formation according to bacterial growth in M63 minimal medium. According to previous publication, specific biofilm formation index of wild type AIEC strain LF82 should be between 2 and 4 (Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013).
Biofilm formation assay on paraformaldehyde-fixed I-407 IEC monolayers
Intestine-407 (I-407) cells were derived from human intestinal embryonic jejunum and ileum tissues. They were maintained in an atmosphere containing 5% CO2 at 37 °C in Minimum Essential Medium supplemented with 10% (vol/vol) fetal bovine serum; 1% of nonessential amino acids; 1% of L-glutamine; 1% of Penicillin/Streptomycin/Amphotericin B solution and 1% of vitamin mix.
Monolayers were seeded on coverslip in 24-well tissue culture plates with 4 x 105 cells per well and incubated for 20 h at 37 °C with 5% CO2.
Monolayers were then washed once with 1ml PBS, fixed for 15 min in 4% formaldehyde (stock solution diluted in sterile PBS) at room temperature without agitation, and then washed 4 times with PBS.
Bacterial strains expressing green fluorescent protein (GFP) (Valdivia et al., 1996) were prepared as previously described above, from step A1 to A2, in M63 minimal medium supplemented with 1 mM of MgSO4 and 8 g.L-1 glucose.
1 ml of this bacterial suspension was applied on the surface of fixed I-407 cell monolayers, and incubated overnight at 30 °C without shaking.
The liquid was removed by decantation, and the wells were washed 3 times using 1 ml of sterile PBS without incubation.
I-407/biofilm complex was then fixed for 15 min using 4% formaldehyde.
Phalloidin-tetramethyl rhodamine isocyanate was used to visualize actin and Hoechst 33258 was used to visualize nuclei. For this purpose, Phalloidin-tetramethyl rhodamine isocyanate was diluted in sterile PBS at a final concentration of 1μg/ml and Hoechst 33258 was added at a final concentration of 1μg/ml. This solution was added to the wells and incubated for 20 min at room temperature. After incubation, the wells were washed 5 times using 1 ml of sterile PBS without incubation.
Coverslips containing the fixed and stained I-407/biofilm complex were then removed from the 24-well plate and mounted on slide using Vectashield.
The slides were examined with a confocal microscope. An example of biofilm formation on paraformaldehyde-fixed I-407 IEC monolayers is presented Figure 1.
Images from biofilm formation assay at the surface of intestinal epithelial cells I-407 monolayers could be analyzed for thickness and/or roughness with the computer program COMSTAT1 (Haydorn et al., 2000).
Figure 1. Confocal analysis of biofilm formation of a poor biofilm producer (Non pathogenic Escherichia coli K12 strain C600 (A) and a strong biofilm producer (Adherent-Invasive Escherichia coli strain LF82 (B) at the surface of a PFA-fixed monolayer of I-407 intestinal epithelial cells. Bacteria express GFP (green), actin is stained in red using phalloidin-TRITC, and DNA is stained in blue using Hoechst. Bars, 50 m.
Notes
Due to high variation observed with both of these biofilm formation assays, they need to be performed at least in triplicate, including controls.
Recipes
Minimum Essential Medium supplemented with
10% (vol/vol) fetal bovine serum
1% of nonessential amino acids
1% of L-glutamine
1% of Penicillin/Streptomycin/Amphotericin B solution
1% of vitamin mix
Acknowledgments
This study was supported by the Ministère de la Recherche et de la Technologie, the Institut National de la Santé et de la Recherche Médicale and the Université d’Auvergne (UMR Inserm 1071), the Institut National de la Recherche Agronomique (USC INRA 2018) and by grants from the Association F. Aupetit (AFA). We thank the CICS platform for confocal microscopy. This protocol is adapted from previously published papers (Danese et al., 2000; Naves et al., 2008; Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013). This work is dedicated to Arlette Darfeuille-Michaud.
References
Chassaing, B. and Darfeuille-Michaud, A. (2011). The commensal microbiota and enteropathogens in the pathogenesis of inflammatory bowel diseases. Gastroenterology 140(6): 1720-28.
Chassaing, B. and Darfeuille-Michaud, A. (2013). The sigmaE pathway is involved in biofilm formation by Crohn's disease-associated adherent-invasive Escherichia coli. J Bacteriol 195(1): 76-84.
Danese, P. N., Pratt, L. A., Dove, S. L. and Kolter, R. (2000). The outer membrane protein, antigen 43, mediates cell-to-cell interactions within Escherichia coli biofilms. Mol Microbiol 37(2): 424-432.
Darfeuille-Michaud, A., Neut, C., Barnich, N., Lederman, E., Di Martino, P., Desreumaux, P., Gambiez, L., Joly, B., Cortot, A. and Colombel, J. F. (1998). Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease. Gastroenterology 115(6): 1405-1413.
Martinez-Medina, M., Naves, P., Blanco, J., Aldeguer, X., Blanco, J. E., Blanco, M., Ponte, C., Soriano, F., Darfeuille-Michaud, A. and Garcia-Gil, L. J. (2009). Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC). BMC Microbiol 9: 202.
Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K. and Molin, S. (2000). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146 ( Pt 10): 2395-2407.
Naves, P., del Prado, G., Huelves, L., Gracia, M., Ruiz, V., Blanco, J., Dahbi, G., Blanco, M., Ponte Mdel, C. and Soriano, F. (2008). Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains. Microb Pathog 45(2): 86-91.
Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L. and Falkow, S. (1996). Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. Gene 173(1 Spec No): 47-52.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Chassaing, B. and Darfeuille-Michaud, A. (2013). Adherent-invasive Escherichia coli Biofilm Formation Assays. Bio-protocol 3(23): e982. DOI: 10.21769/BioProtoc.982.
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Category
Microbiology > Microbial biofilm > Biofilm culture
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983 | https://bio-protocol.org/en/bpdetail?id=983&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Chitinase Assay from Cultured Bone Marrow Derived Macrophages
DW Danielle Worth
JN J. Philip Nance
Emma H. Wilson
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.983 Views: 7705
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS Pathogens Nov 2012
Abstract
Chitinases are chitin-degrading enzymes. Chitinases play essential roles in combating chitin-containing pathogens as well as established roles in asthmatic inflammation. This assay is designed to detect chitinase activity in macrophage cell lysates. The chitin substrate is labeled with 4-methylumbelliferone. Hydrolysis of chitin releases 4-methylumbelliferone, and is measured fluorometrically to determine chitinase activity.
Materials and Reagents
Cells to be analyzed. e.g. Bone Marrow-derived macrophages from C57BL/6 mice
L-cell
cDMEM/F12
4-Mehtylumbelliferyl labeled substrates:
4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (exochitinase activity; β–N-acetylglucosaminidase activity) (Sigma-Aldrich, catalog number: M2133 )
4-Methylumbelliferyl N,N′-diacetyl-β-D-chitobioside (exochitinase activity; chitobiosidase activity) (Sigma-Aldrich, catalog number: M9763 )
4-Methylumbelliferyl β-D-N,N′,N′′-triacetylchitotriose (endochitinase activity) (Sigma-Aldrich, catalog number: M5639 )
Chitinase from Trichoderma viride (Sigma-Aldrich, catalog number: C6242 )
Methylumbelliferone Standard (50 mg/ml) (Sigma-Aldrich, catalog number: M1381 )
PBS
DMSO
Plate reader
Diabasic Sodium Phosphate
Citric Acid
Glycine
DTT
Assay buffer (see Recipes)
Stop buffer (see Recipes)
Protein lysis buffer (see Recipes)
Substrate Stock (see Recipes)
Positive control (see Recipes)
Equipment
Flat bottom black 96-well plates
Fluorescent plate reader (Molecular Devices)
Procedure
Cells to be analyzed. We have used Bone Marrow-derived macrophages from C57BL/6 mice.
In brief, bone marrow is flushed from tibias and femurs of 6-8 week old mice with 10 ml complete DMEM/F12 supplemented with 20% L-cell supernatant (day 0).
Bone marrow cells are plated in non tissue culture treated 10 cm petri dishes ~10 ml cells per dish. After 4 days, add 10 ml additional media.
Cells are harvested by gentle scraping on day 7.
Macrophages are plated at 50,000 cells/well in 96 well plates in cDMEM/F12 in 10% L-cell supernatant and allowed to rest for 3 days.
On day 10, media is changed to cDMEM/F12 without L-cell supernatant and rested overnight.
Cells are ready for use on day 11.
Aspirate media in each well from the culture plates, and add lysis buffer to each (50 μl for 96 well plate, and 200 μl for a 24 well plate). Place the plate on the rocker for 15 min at room temperature.
Dilute an aliquot of 4-Methylumbelliferyl substrate stock solution 40-fold in assay buffer, such that the final concentration of the substrate is 0.5 mg/ml. This will be termed the “working solution”.
Approximately 100 μl of working solution will be needed per sample. Allow solution to equilibrate in 37 °C water bath.
For each form of chitinase being tested there is a unique substrate. Separate assay plates will needed for each substrate.
Prepare Methylumbelliferone standards by diluting the top Methylumbelliferone standard 1:100 (500 μg/ml), 1:1,000 (50 μg/ml), and 1:10,000 (5 μg/ml) in stop buffer.
For best resolution, add 2 μl top Methylumbelliferone standard to 198 μl of stop buffer for the 1:100 dilution, followed by a 1:10 dilution series.
Samples will be diluted further in the assay plate to yield 1,000 ng, 500 ng, 100 ng, and 10 ng (Figure 1).
Load the standard wells in triplicate to the flat bottom black 96-well plate as demonstrated in Figure 1.
Figure 1. Plate set-up for chitinase assay. Red wells indicate 4-Methylumbelliferone standards, Purple indicates the sample blank (Working Solution), Green represents the positive control, and blue wells indicate sample wells.
Dilute the chitinase positive control 1:200 in PBS to yield a final concentration of 1 μg/ml and load positive control wells with 10 μl chitinase and 90 μl working solution
Use 100 μl working solution as the sample blank
Add 90-99 μl working solution to each sample well in triplicate followed by 1-10 μl of each sample (samples are in lysis buffer)
The amount of sample used for this assay will need to be optimized. Some samples may contain so much chitinase activity that the fluorescence will be saturated. This will vary considerably depending on the amount of cells plated and the chitinase activity of those cells.
Wrap plate in foil and incubate at 37 °C for 30 min
Incubation time may also need to be optimized. Cell lysates with high chitinase activity can be incubated for as little as 15 min. Alternatively, samples may be incubated for up to 1 h.
Add 200 μl of stop buffer to each well to stop the reaction.
Fluorescence can be measured on a plate reader at an excitation of 360 nm and emission of 450 nm within 30 min.
Chitinase activity is calculated from the standard curve. Alternatively, chitinase activity may be calculated using the following equation:
Units/mL= (Fluoresencesample – Fluoresenceblank) x 1.9 x 0.3 x Dilution Factor
Fluoresence100 ng standard x reaction time x sample Volume
Recipes
Assay buffer
Phosphate-Citrate Buffer pH=5.2 (26.7 ml of 0.2 M diabasic Sodium Phosphate, 23.3 ml of 0.1 M Citric Acid, top up to 100 ml DI water)
Stop buffer
Glycine-NaOH buffer pH = 10.6 (combine 25 ml 0.2 M glycine stock solution with 22.75 ml 0.2 M NaOH, and dilute with DI water to make a 100 ml solution)
Protein lysis buffer
50 mM Tris HCl (pH 7.5)
200 mM NaCl
10% Glycerol
0.5% TX-100
1 mM DTT (added to buffer fresh, just before adding to cultures)
Substrate Stock
Prepare 20 mg/ml 4-Methylumbelliferyl substrate in DMSO.
Positive control
Prepare 0.2 mg/ml chitinase from Trichoderma in PBS.
Acknowledgments
This protocol is adapted from Nance et al. (2012).
References
Nance, J. P., Vannella, K. M., Worth, D., David, C., Carter, D., Noor, S., Hubeau, C., Fitz, L., Lane, T. E., Wynn, T. A. and Wilson, E. H. (2012). Chitinase dependent control of protozoan cyst burden in the brain. PLoS Pathog 8(11): e1002990.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Worth, D., Nance, J. P. and Wilson, E. H. (2013). Chitinase Assay from Cultured Bone Marrow Derived Macrophages. Bio-protocol 3(23): e983. DOI: 10.21769/BioProtoc.983.
Nance, J. P., Vannella, K. M., Worth, D., David, C., Carter, D., Noor, S., Hubeau, C., Fitz, L., Lane, T. E., Wynn, T. A. and Wilson, E. H. (2012). Chitinase dependent control of protozoan cyst burden in the brain. PLoS Pathog 8(11): e1002990.
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Category
Immunology > Immune cell function > Macrophage
Biochemistry > Protein > Activity
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Preparation of Primary Neurons from Rat Median Preoptic Nucleus (MnPO)
Emmanuelle Berret
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.984 Views: 9816
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Feb 2013
Abstract
Studying cell physiology is really important to understand their function and to determine action mechanisms taking place in these cells. Using brain slices can be sometime difficult to directly access to cells like neurons. Even if it more steps are needed to get cultured cells, this type of preparation allow a better access to neurons and provide a good way to study their basal properties. Here we describe a protocol of a short term primary neurons culture from MnPO, allowing to kept neurons in good condition to perform electrophysiological recordings.
Materials and Reagents
Wistar rats (3 weeks old) (Charles River Laboratories International)
Ketamine-xylasine mixture
Pronase (Sigma-Aldrich, catalog number: 9036-06-0 )
Thermolysin (Sigma-Aldrich, catalog number: 9073-78-3 )
Bovine Serum Albumin (BSA) (Life Technologies, catalog number: 15561020 )
Laminin (Sigma-Aldrich, catalog number: 114956-81-9 )
Ruthenium red
KCl (Sigma-Aldrich, catalogue number: 7447-40-7 )
CaCl2 (Sigma-Aldrich, catalogue number: 10043-52-4 )
MgCl2 (Sigma-Aldrich, catalogue number: 7786-30-3 )
NaHCO3 (Sigma-Aldrich, catalogue number: 144-55-8 )
NaH2PO4 (Sigma-Aldrich, catalogue number: 7558-80-7 )
NaCl (Sigma-Aldrich, catalogue number: 7647-14-5 )
Sucrose (Sigma-Aldrich, catalogue number: 57-50-1 )
HEPES (Sigma-Aldrich, catalogue number: 7365-45-9 )
D-Glucose (Sigma-Aldrich, catalogue number: 50-99-7 )
Dissection solution (see Recipes)
Artificial Cerebrospinal Fluid (aCSF) (see Recipes)
Equipment
Bath
Vibratome
0.45 μm sterilize filter
350 μm Sagittal slice containing MnPO
Needle
Pasteur pipettes
Centrifuge
37 °C, 95% O2-5% CO2 Cell culture incubator
6 hole petri dish (Thermo Fisher Scientific, catalog number: 50-341-84 )
Micro-cover glasses (Thermo Fisher Scientific, catalogue number: NC9216450 )
15 ml Falcon tubes
Oxygen and Carbogen tank
Binocular
Procedure
Micro-cover glass is coated with laminin (5 μg/ml) and incubated at 37 °C, 95% O2-5% CO2 Cell culture incubator, 3 h before culture preparation.
Wistar rats is deeply anesthetized by intraperitoneal injection of a ketamine-xylasine mixture (87.5 and 12.5 ml/kg, respectively) and decapitated.
The brain is removed from the skull and immersed in oxygenated (95% O2-5% CO2) ice-cold (2 °C) dissection solution. Oxygenation system consist on a 95% O2-5% CO2 tank connected to a bath with a line transmitting a gentle bubbling. Then the brain is fixed with glue on a plate which will be place in the Vibratome tub containing oxygenated dissection solution.
A sagittal slice of 350 μm containing the MnPO is prepared using Vibratome (Figure 1).
Figure 1. Use Vibratome to do brain slices
Using binocular, the ventral region of the MnPO is punched (about 3mm in diameter) out with a curve needle (Figure 2), and placed in 1 ml aCSF solution (in falcon) containing 0.1 mg/ml of pronase for 10 min at 37 °C and oxygenated.
Note: Curve needle is homemade from an aluminum needle tip 3 mm diameter, gently curved at 2/3 of length with a soldering and fixed to a 10 ml syringe.
Figure 2. Use Binocular to realize MnPO micropunch
Micropunch is transferred to a new falcon tube with 1 ml aCSF solution containing 2 mg/ml of BSA for 15 min at 37 °C and oxygenated.
Micropunch is transferred to a new falcon tube with 1 ml aCSF solution containing 0.1 mg/ml of thermolysin for 10 min at 37 °C and oxygenated.
Transfer solution containing tissue in a 1.5 ml Eppendorf tube.
The tissue is then mechanically dissociated by successive trituration with glass Pasteur pipettes (Figure 3).
Note: Use 4 Pasteur pipettes whose diameter has previously been gradually reduced with flame from no change to 50 μm. This step is done at room temperature. The come and go flow should be strong enough to separate cells, but also enough gentle to not break cell membrane. The size of micropunch should reduce from pipette to another, and completely disappear using the last pipette.
Figure 3. Mechanic trituration with Pasteur pipette
Transfer solution containing cells in a 1.5 ml Eppendorf tube.
Solution containing cell is centrifuged at 1500 x g during 2 min at room temperature.
Note: Sometime pellet is really small and difficult to see, but there are cells on the wall of the tube.
The supernatant is removed and cells are re-suspended in 50 μl aCSF.
Note: Slow agitation for re-suspension to not break the membranes.
The aCSF solution containing cells (50 μl) is directly platted on micro cover glasses beforehand treated with laminine, then disposed in 6 holes petri dish, and incubated 1 h at 37 °C, 95% O2-5% CO2, before patch-clamp recording.
Notes:
There is no culture media, cells remains during 1 h in aCSF solution. Do not exceed 1 h of incubation. Density of cell is relatively low, about 100 cells on 1 micro cover glass.
Be sure that the solution stays on the micro-cover glass in bead, if not preparation will be dry and unusable.
Recipes
Dissection solution
200 mM sucrose
10 mM D-Glucose
2 mM KCl
1 mM CaCl2
3 mM MgCl2
26 mM NaHCO3
1.25 mM NaH2PO4, pH 7.4
Adjusted with CO2 bubbling and verified with Ruthenium red
Filter sterilize (0.45 μm)
Stored at 4 °C
Artificial cerebrospinal fluid/aCSF
140 mM NaCl
3.1 mM KCl
2.4 mM CaCl2
1.3 mM MgCl2
10 mM HEPES
10 mM D-Glucose, pH 7.4 adjusted with KOH, osmolarity 300 MOsm
Filter sterilize (0.45 μm)
Stored at 4 °C
Acknowledgments
This protocol was adapted from: Tremblay et al. (2011). This work was supported by Canadian Institutes of Health Research Grant MOP-178002.
References
Berret, E., Nehme, B., Henry, M., Toth, K., Drolet, G. and Mouginot, D. (2013). Regulation of central Na+ detection requires the cooperative action of the NaX channel and α1 Isoform of Na+/K+-ATPase in the Na+-sensor neuronal population. J Neurosci 33(7): 3067-3078.
Tremblay, C., Berret, E., Henry, M., Nehme, B., Nadeau, L. and Mouginot, D. (2011). Neuronal sodium leak channel is responsible for the detection of sodium in the rat median preoptic nucleus. J Neurophysiol 105(2): 650-660.
Article Information
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© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Cell isolation and culture > Cell isolation
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Burkholderia glumae Competent Cells Preparation and Transformation
Hiromasa Saitoh
RT Ryohei Terauchi
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.985 Views: 11469
Reviewed by: Tie Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Plant Journal May 2013
Abstract
Bukholderia glumae is a gram-negative bacterium which causes grain rot, seedling rot and panicle blight in rice and bacterial wilt in many field crops. This bacterium has been reported from major rice growing regions around the world and is now considered as an emerging major pathogen of rice (Tsushima et al., 1996; Jeong et al., 2003; Kim et al., 2010; Ham et al., 2011). Here we describe two methods for competent cells preparation and transformation of B. glumae. Using these methods, we have applied effector detector system (Sohn et al., 2007) to B. glumae (Sharma et al., 2013).
Keywords: Burkholderia glumae Competent cells Transformation PEDV5 based vectors
Materials and Reagents
Burkholderia glumae strain 106619 (National Institute of Agricultural Sciences Genebank, Tsukuba, Ibaraki, Japan)
pEDV5 based vectors (Fabro et al., 2011; Sharma et al., 2013)
Gentamycin
Tryptone
Yeast extract
Protease peptone no. 3
Ice
Lysogeny Broth (LB) medium (see Recipes)
King’s Broth (KB) agar (see Recipes)
KB agar with 25 ng/ml gentamycin (see Recipes)
10% glycerol (see Recipes)
300 mM sucrose solution (see Recipes)
Equipment
Deep freezer
Autoclave
Clean bench
Petri plates
Sterile 1.5 ml tubes
Sterile 15 ml tubes
Sterile 50 ml tubes
Incubation Shaker
Aluminum foil
Spectrophotometer
Centrifuge
Toothpick
MicroPulser/Gene Pulser Cuvettes, 0.2 cm gap (Bio-Rad Laboratories, catalog number: 165-2089 )
MicroPulser/Gene Pulser Cuvettes, 0.1 cm gap (Bio-Rad Laboratories, catalog number: 165-2086 )
Gene Pulser XcellTM Electroporation Systems (Bio-Rad Laboratories)
Minisart filters (pore size 0.2 μm) (Sigma-Aldrich, catalog number: 16534K )
Disposable Cell Spreaders
Procedure
Part I: Conventional method
Preparation of competent cells
10 μl of glycerol stock of the B. glumae strain is inoculated to 20 ml of LB medium in a 50 ml tube and further incubated at 28 °C for 16-40 h with horizontal shaking at 200 rpm until OD600 = 0.8 is achieved.
The lid of the tube is opened for 30 sec under a clean bench.
The tube is incubated again at 28 °C for 4 h with horizontal shaking at 200 rpm.
The tube is centrifuged twice at 800 x g at 4 °C for 5 min.
Each time the pellet is dissolved in 20 ml of autoclaved cold 10% glycerol.
The pellet is dissolved in 200 μl of cold 10% glycerol and divided into 50 μl aliquots and stored at -80 °C for a further transformation step.
Transformation
Remove from -80 °C tubes containing 50 μl of electro-competent B. glumae cells.
Thaw the cells on ice.
Add ~1 μg of pEDV5 based vector into the B. glumae cells. Incubate on ice up to 3 min.
Transfer the mixture of cells + DNA to a cold electroporation cuvette (0.2 cm electrode gap). Make sure the suspension is at the bottom of the cuvette.
Set the Gene Pulser apparatus at 25 μF the volt at 2.5 kV. Set the Pulse resistance controller to 200 ohms.
Place the cold cuvette in the chamber slide (Cuvette notch facing away from you).
Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
Electroporate by pushing the red button.
Remove the cuvette from the chamber and immediately add 1 ml of LB medium to the cuvette and quickly resuspend the cells by pipetting.
Transfer the cell suspension from the cuvette to 1.5 ml tubes and incubate on shaker (200 rpm) at 28 °C for 2 h to allow recovery and expression of the gentamycin resistance marker (Clean cuvettes successively with dH2O, EtOH, sterile water, then wrap with aluminum foil then autoclave).
Pipette 200 μl of each transformation on KB agar plates containing 25 ng/ml gentamycin.
Spread the cells with cell spreaders. Place plates inverted at 28 °C for 2-3 days in the dark.
Part II. High competency method
Preparation of competent cells
A frozen glycerol stock of the B. glumae strain is picked with a toothpick and spread on a KB agar plate.Place plates inverted at 28 °C for 2-3 days in the dark.
Freshly grown B. glumae colony is inoculated to 5 ml of LB medium in a 15 ml tube and further incubated at 28 °C for 16 h with horizontal shaking at 200 rpm.
1 ml aliquots is centrifuged twice at 3,500 x g at 4 °C for 5 min.
Each time the pellet is dissolved in 1 ml of filter sterilized and room temperature 300 mM sucrose solution.
The pellet is dissolved in 200 μl of 300 mM sucrose solution and divided into 100 μl aliquots and used the cells immediately for a further transformation step.
Transformation
Add ~1 μg of pEDV5 based vector into the B. glumae cells.
Transfer the mixture of cells + DNA to an electroporation cuvette (0.1 cm electrode gap). Make sure the suspension is at the bottom of the cuvette.
Set the Gene Pulser apparatus at 25 μF the volt at 1.8 kV. Set the Pulse resistance controller to 200 ohms.
Place the cuvette in the chamber slide (Cuvette notch facing away from you).
Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
Electroporate by pushing the red button.
Remove the cuvette from the chamber and immediately add 1 ml of LB medium to the cuvette and quickly resuspend the cells by pipetting.
Transfer the cell suspension from the cuvette to 1.5 ml tubes and incubate on shaker (200 rpm) at 28 °C for 2 h to allow recovery and expression of the gentamycin resistance marker (Clean cuvettes successively with dH2O, EtOH, sterile water, then wrap with aluminum foil then autoclave).
Pipette 200 μl of each transformation on KB agar plates containing 25 ng/ml gentamycin.
Spread the cells with cell spreaders. Place plates inverted at 28 °C for 2-3 days in the dark.
Recipes
LB medium
Mix 5 g of tryptone
2.5 g of yeast extract
5 g of NaCl with 800 ml dH2O
Add 0.2 ml of 5 N NaOH
Add dH2O to 1,000 ml and autoclave it for 20 min
KB agar
Mix 20 g of protease peptone no. 3
10 g of glycerol
1.5 g of K2HPO4
1.5 g of MgSO4.7H2O with 800 ml dH2O
Adjust to pH 7.2
Add dH2O to 1,000 ml, add 15 g of agar and autoclave it for 20 min
KB agar with 25 ng/ml gentamycin
Cool down the autoclaved KB agar to 50 °C
Add 1 ml of 25 mg/ml gentamycin
Pour in 90 mm x 15 mm Perti dish
10% glycerol
Mix 100 g of glycerol with 900 ml dH2O and autoclave it for 20 min
300 mM sucrose solution
Mix 10.27 g of sucrose with 80 ml
Add dH2O to 100 ml and filter (0.2 μm) sterilize it
Acknowledgments
This protocol was adapted from Sharma et al. (2013). This work was supported by the ‘Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN)’ (Japan) and Japan Society for the Promotion of Science (JSPS) grants no. 18310136 and 20380027, and ‘The Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation PMI-0010)’ and the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research on Innovative Areas 23113009); and by JSPS grant nos 22780040 and 2301518 to H. Saitoh, JSPS grant no. 2200214 to R. Terauchi. The financial assistance received from JSPS to Shailendra Sharma and Shiveta Sharma for carrying out this study is gratefully acknowledged. S. Kamoun and J.D.G. Jones were supported by The Gatsby Foundation (United Kingdom).
References
Ham, J. H., Melanson, R. A. and Rush, M. C. (2011). Burkholderia glumae: next major pathogen of rice? Mol Plant Pathol 12(4): 329-339.
Jeong, Y., Kim, J., Kim, S., Kang, Y., Nagamatsu, T. and Hwang, I. (2003). Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops. Plant Disease 87(8): 890-895.
Kim, J., Kang, Y., Kim, J.G., Choi, O. and Hwang, I. (2010). Occurrence of Burlkholderia glumae on rice and field crops in Korea. Plant Pathol J 26(3): 271-272.
Sharma, S., Sharma, S., Hirabuchi, A., Yoshida, K., Fujisaki, K., Ito, A., Uemura, A., Terauchi, R., Kamoun, S., Sohn, K. H., Jones, J. D. and Saitoh, H. (2013). Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells. Plant J 74(4): 701-712.
Sohn, K. H., Lei, R., Nemri, A. and Jones, J. D. (2007). The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana. Plant Cell Online 19(12): 4077-4090.
Tsushima, S., Naito, H. and Koitabashi, M. (1996). Population dynamics of Pseudomonas glumae, the causal agent of bacterial grain rot of rice, on leaf sheaths of rice plants in relation to disease development in the field. Ann Phytopathol Soci Japan 62(2): 108-113.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Microbiology > Microbe-host interactions > In vivo model
Plant Science > Plant immunity > Perception and signaling
Molecular Biology > DNA > Transformation
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ATP and Lactate Quantification
Isaline Rowe
Marco Chiaravalli
AB Alessandra Boletta
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.986 Views: 16258
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Original Research Article:
The authors used this protocol in Nature Medicine Apr 2013
Abstract
Cells use glucose to generate energy by two different metabolic processes: lactic fermentation and aerobic respiration. In the first common series of reactions, glucose is converted into pyruvate. In anaerobic conditions, pyruvate is transformed into lactate, this process yields to 2 ATP molecules per glucose molecule. In the presence of oxygen, pyruvate is imported into mitochondria where it is used in the Krebs (or TCA) cycle and oxydative phosphorylation. The global process of oxydative phosphorylation yields to 32 ATP per glucose molecule. For reasons not fully understood, in some pathological cases like cancer, cells use anaerobic glycolysis even in the presence of oxygen, in which case the process is called aerobic glycolysis (or Warburg effect). This results in an increased uptake of glucose and lactate production. Measure of intracellular ATP content and lactate concentrations can provide a readout of aerobic glycolyis.
Materials and Reagents
For intracellular ATP content evaluation
Murine Embrionic Fibroblasts (MEF)
Dulbecco’s Modified Eagle’s Medium High glucose with L-glutamine (DMEM) (Gibco®, catalog number: 11965 )
Foetal Calf Serum (FCS) (Gibco®, catalog number: 16010 )
Penicillin/Streptamycin (Gibco®, catalog number: 15070 )
Phosphate Buffered Saline (PBS) (EuroClone, catalog number: E0B4004L )
Protein Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
Bovine Serum Albumine (BSA) (Sigma-Aldrich, catalog number: A4503 )
NaCl
Na4HPO4/NaH2PO4
Glycerol
Triton X-100
Complete protease inhibitors (Roche Diagnostics, catlalog number: 11697498001 )
Phosphatase inhibitors (1 mM final concentration of glycerophosphate, sodium orthovanadate and sodium fluoride)
Complete protease inhibitors (Roche Diagnostics)
ATP détermination kit (Life Technologies, catalog number: A22066 )
Lysis buffer (see Recipes)
Standard Reaction Solution (see Recipes)
For Lactate quantification
MEF
EnzyChromTM L-lactate Assay Kit (BioAssay Systems, catalog number: ECLC-100 )
Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Gibco®, catalog number: 11965)
Foetal Calf Serum (FCS) (Gibco®, catalog number: 16010 )
Penicillin/Streptamycin (Gibco®, catalog number: 15070 )
0.4% Trypan Blue stain (Life Technologies, catalog number: T10282 )
Equipment
For intracellular ATP content evaluation
6 wells plates (Corning, Costar®, catalog number: CLS 3506 )
Cell Scraper (Nunc®, catalog number: 179707 )
Luminometer Gloomax 20/20
Spectrophotometer
37 °C 5% CO2 Cell culture incubator
Centrifuge spinning speed: 15682,186 G-force (13,000 rpm, 8,3 cm radius)
For Lactate quantification
37 °C 5% CO2 Cell culture incubator
6 wells plates (Corning, Costar®, catalog number: CLS 3506)
96 wells plates (Corning, Costar®, catalog number: CLS 3596 )
Countess automated cell counter with countess Cell Counting Chamber Slides (Life Technologies)
Plate reader at 565 nm
Procedure
For intracellular ATP content evaluation
MEF are plated at 60-70% cell confluency.
Cells are washed twice with PBS
Cells are lysed with a cell scraper in 35 μl of lysis buffer containing phosphatase inhibitors (1 mM final concentration of glycerophosphate, sodium orthovanadate and sodium fluoride) for 30 min at 4 °C.
Centrifuge at 13,000 rpm for 15 min at 4 °C.
Supernatant is isolated.
The sample is diluted at 1:1,000 (1 μl of sample is added to 1,000 μl of Biorad solution) in a Biorad solution (200 μl of Biorad and 800 μl of water).
Optical Density (OD) is read at the spectrophotometer at 595 nm.
The concentration of the sample is determined using a standard curve from BSA.
Samples are diluted at a final concentration of 25 ng/ml.
250 ng of total proteins in parallel of the ATP standard curve (0 nM, 1 nM, 10 nM, 100 nM, 1,000 nM of ATP) in 10 μl is added to 90 μl the Standard Reaction Solution.
Luciferase activity is measured with a luminometer.
ATP is quantified for each sample using the ATP standard curve.
Lactate quantification
Cells are plated in 6 wells plates with 3 ml of medium (10% DMEM-FCS-1% Penicillin/Streptamycin).
The medium is changed when the cells reach 100% cell confluence.
The medium is collected after 24 h.
Cells are trypsinized and alive cells are counted with Trypan blue 1:1.
The medium collected is centrifuged at 13,000 rpm for 10 min
The supernatant (clean medium) is diluted 1:10 in DMEM.
20 μl of each sample and of the standard curve (0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1 mM of Lactate in DMEM) is put in wells of a 96 wells plate.
80 μl of assay buffer from EnzyChrom L-Lactate Assay Kit is added in each well.
Mix well (Pipetting up and down).
D0 is absorbed 565 nm at the beginning of the incubation ( t0) and after 20 min (t20).
The difference of absorbtion (delta OD) is determined for the samples and the standard curve.
The concentration of lactate for each sample is obtained from the standard curve.
The quantity of lactate is normalized by the final number of cells.
Recipes
Lysis buffer
150 mM NaCl
20 mM Na4HPO4/NaH2PO4
10% glycerol
1% Triton X-100 (pH 7.2)
Complete protease inhibitors
Standard Reaction Solution
1x Reaction Buffer
0.1 mM DTT (dithiothreitol)
0.5 ml 0.5 mM of D-luciferine
2.5 μg/ml firefly luciferase and incubated at room temperature for 15 min (in the dark)
Acknowledgments
We thank all the co-authors of the article: Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y. and Musco, G. and the other members of the lab Boletta.
References
Rowe, I., Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y., Musco, G. and Boletta, A. (2013). Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med 19(4): 488-493.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Rowe, I., Chiaravalli, M. and Boletta, A. (2013). ATP and Lactate Quantification. Bio-protocol 3(23): e986. DOI: 10.21769/BioProtoc.986.
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Category
Cell Biology > Cell metabolism > Carbohydrate
Biochemistry > Carbohydrate > Lactate
Cell Biology > Cell signaling > Respiration
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987 | https://bio-protocol.org/en/bpdetail?id=987&type=0 | # Bio-Protocol Content
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Mitochondrial Transmembrane Potential (ψm) Assay Using TMRM
Isaline Rowe
AB Alessandra Boletta
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.987 Views: 27548
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Original Research Article:
The authors used this protocol in Nature Medicine Apr 2013
Abstract
During cellular respiration, nutrients are oxidized to generate energy through a mechanism called oxidative phosphorylation, which occurs in the mitochondria. During oxidative phosphorylation, the gradual degradation of molecules through the TCA cycle releases electrons from the covalent bonds that are broken. These electrons are captured by NAD+ through its reduction into NADH. Finally, NADH transports the electrons to the complexes of the electron chain in the internal membrane of mitochondria. These complexes use the energy released by the electrons to pump protons into the intermembrane space, generating an electrochemical gradient across the internal membrane of mitochondria, which provides energy for the ATP-synthase complex, ultimately producing adenosine triphosphate (ATP). We assessed the mitochondrial membrane potential (ψm) using tetramethylrhodamine methyl ester (TMRM), a cell-permeant, cationic, red fluorescent dye. To measure specifically the mitochondrial membrane potential (ψm) we quantified the fluorescence intensity before and after applying FCCP, a mitochondrial electron chain uncoupler. The difference of intensity before and after applying FCCP corresponds specifically to the mitochondrial membrane potential. We analyzed mitochondrial membrane potential (ψm) by cytofluorimetry. The ratio between the total level of signal and the signal generated after uncoupling provided a normalized value for the difference in cell size. Furthermore, to normalize for the different size of cells that we were analyzing we have analyzed TMRM in live imaging using IN Cell Analyzer, so that the level of mitochondrial membrane potential could be detected per unit of mitochondrial membrane area measured. Thus, our protocol can also be used to compare the mitochondrial membrane potential of cells that are different in size.
Materials and Reagents
Murine Embrionic Fbroblasts (MEF)
Phenol-red free HBSS (Gibco®, catalog number: 14175-079 )
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gibco®, catalog number: 15630-080 )
Tetramethylrhodamine methyl ester (TMRM) (Life Technologies, catalog number: T668 )
Cyclosporin H (Enzo Life Sciences, catalog number: ALX-380-286-M001 )
Hoechst 33342
Carbonylcyanide-p-trifluoromethoxyphenyl hydrazone FCCP (Sigma-Aldrich, catalog number: C2920-10MG )
Trypsin (451) - Trypsin, 0.05% (1X) with 0.53 mM EDTA 4Na, liquid, 20 x 100 ml
(Gibco®, catalog number: 25300096 )
Equipment
IN Cell Analyzer 1000 (General Electric Company)
37 °C 5% CO2 Cell culture incubator
12 well pates (Corning, Costar®, catalog number: CLS 3513 )
FACScan (BD)
Centrifuge spinning speed: 15,682,186 x g (G-force) (13,000 rpm, 8,3 cm radius)
Software
IN Cell Investigator Analysis software (General Electric Company)
BD CellQuest software
FlowJo software
Procedure
For quantitative real-time analysis
Cells are plated at 50% cell confluence.
Cells are incubated for 30 min at 37 °C in phenol-red free HBSS with 10 mM HEPES, 20 nM TMRM, 2 μM cyclosporine-H, inhibitor of multidrug resistance pump activity but not of the permeability transition pore (multi drug resistance pump activity can affect the mitochondrial lloading with TMRM) and 2 μg/ml Hoechst 33342, nuclear dye that can be used in living cells.
Sequential images were taken before (3 time points) and after 4 μM FCCP (3 time points) was injected in a motorized way for TMRM (535 nm excitation filter; 600 nm emission) and Hoechst 33342 (360 nm excitation filter; 460 nm emission) in different fields (4 fields acquired for each well and 6 wells for each experimental condition) every 3 min with an IN CELL Analyzer 1000.
Images are automatically analyzed with IN Cell Investigator Analysis software to measure the TMRM intensity. Fluorescence intensity is measured on the average of 10 points randomly selected for each field at the first and the last time point, the background corresponding to an area without cell is removed for each field.
The fluorescence intensity measured at the last time point (after applying FCCP) is normalized by the fluorescence intensity measured at the first time point of the experiment.
For FACS analysis
Cells are plated at 50% confluence.
Cells are trypsinized (at least 2 millions of cells).
Cells are centrifuged for 5 min at G-force 13,62.
Cells are washed in PBS, resuspended in phenol-red free HBSS with 10 mM HEPES and counted.
Cells (300,000 cells per tube, each condition done in triplicate) are resuspended the in phenol-red free HBSS with 10 mM HEPES and 20 nM TMRM in the presence of 2 μM of the multidrug resistance pump inhibitor cyclosporine-H and incubated for 30 min at 37 °C. In parallel, cells are incubated 5 min in the same conditions with an uncoupling agent, 4 μM FCCP, to measure the specific mitochondria staining.
FACS analysis for TMRM was performed on BD FACScan with CellQuest software and analysed using FlowJo software. Fluorescence intensity of TMRM was calculated by gating on live cells.
The intensity of the fluorescence with FCCP was normalized by the intensity without FCCP in order to rule out the possibility that the difference of the intensity is only or mainly caused by the cell sizes.
Acknowledgments
We thank all the co-authors of the article: Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y. and Musco, G., the other members of the lab Boletta, Casari, G. and Cassina, L. and the San Raffaele microscopy facility (Alembic).
References
Rowe, I., Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y., Musco, G. and Boletta, A. (2013). Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med 19(4): 488-493.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Rowe, I. and Boletta, A. (2013). Mitochondrial Transmembrane Potential (ψm) Assay Using TMRM. Bio-protocol 3(23): e987. DOI: 10.21769/BioProtoc.987.
Download Citation in RIS Format
Category
Cell Biology > Cell signaling > Respiration
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988 | https://bio-protocol.org/en/bpdetail?id=988&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Determination of the Intracellular Calcium Concentration in Peritoneal Macrophages Using Microfluorimetry
SG Silvia González-Ramos
C Luz María G. Carrasquero
ED Esmerilda G. Delicado
MM María T. Miras-Portugal
MF María Fernández-Velasco
LB Lisardo Boscá
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.988 Views: 17175
Reviewed by: Fanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Immunology Apr 2013
Abstract
Calcium is one of the most important intracellular messengers in biological systems. Ca2+ microfluorimetry is a valuable tool to assess information about mechanisms involved in the regulation of intracellular Ca2+ levels in research on cells and in living tissues. In essence, the use of a dye that fluoresces in the presence of a target substance allows the detection of changes in the concentration of this molecule by determining the changes in the fluorescence of the probe (increases or decreases, depending on the nature of the dye used; for a review see Tsien et al. 1985). In this regard, there have been developed two different methodologies to assess intracellular Ca2+ measurements. On the one hand, ratiometric methods are based on the use of a ratio between two fluorescence intensities linked to the physicochemical properties of the probe. This allows correction of artifacts due to bleaching, changes in focus, variations in laser intensity, etc. but makes measurements and data processing more complicated since they require more expensive equipment with the possibility to change the wavelength emission/detection in a rapid way. Some ratiometric Ca2+ indicators are Fura-2 and Indo-1. On the other hand, on binding to Ca2+, indicators used for non-ratiometric measurements show a shift in their fluorescence intensity (the free indicator has usually a very weak fluorescence). Therefore, although an increase in fluorescence signal can be related directly to an increase in Ca2+ concentration, the fluorescence intensity depends on many factors such as acquisition conditions, probe concentration, optical path length, balance between the affinity constants of proteins binding Ca2+, among others. However, the fluxes of Ca2+ are of such a magnitude that these interferences are minor contributors to biases in the measurements. There are many non-ratiometric calcium indicators, some of which are Fluo-3, Fluo-4 and Calcium-Green-3. Consequently, the most suitable Ca2+-probe for each experiment will depend on the range of Ca2+ concentration that has to be evaluated, instrumentation, loading requirements, etc. In the present report we describe the protocol employed to quantify intracellular Ca2+ changes in peritoneal macrophages using Fura-2 as a fluorimetric probe and a microfluorimetric protocol that allows quantification of responding cells to a given stimulus, localization of the main intracellular domains sensing Ca2+ changes and a time-resolved analysis of the Fura-2 fluorescence that reflects the intracellular dynamics of Ca2+ in these cells (Través et al., 2013).
Keywords: Calcium fluxes Myeloid cells Single cell analysis Microfluorimetry
Materials and Reagents
Fura 2-AM (Life Technologies)
DMSO (Sigma-Aldrich)
EGTA
CaCl2
HEPES
Trypan Blue
FCS
Vacuum grease
Peritoneal macrophages obtained from Balb/c male mice (8-12 weeks old) 4 days after i.p. administration of 2.5 ml of 3% thioglycollate solution
Note: In our study (Traves et al., 2013), the effect of prostaglandin E2 on the response to P2X/P2Y agonists ATP and BzATP was studied in depth. All these molecules were purchased from Sigma-Aldrich (St. Louis, MO).
10x DPBS without Ca2+ Mg2+ (Lonza)
Locke's solution (see Recipes) (supplemented with 1 mg/ml BSA)
Equipment
Coverslips 0.13-0.16 mm thickness (15 mm of diameter) (SCHOTT AG)
Forceps
35 mm culture dishes
Basic water-jacket CO2 incubator
Perfusion chamber RC-25F (Warner Instruments)
Valves employed for cell perfusion (Warner Instruments)
Perfusion valves controller VC 8 (Warner Instruments)
Vacuum pump
Water bath
NIKON TE-200 microscope with a Plan Fluor x20/0.5 water objective( Nikon Corporation, model:TE-200)
Dichroic mirror (430 nm) and a 510 nm band-pass filter (Omega Optical)
ORCA-ER C4742-80 camera (Hamamatsu Photonics K. K., model: C4742-80)
Filter wheel Lambda 10-2 (Sutter Instrument Company)
Software
MetaFluor 6.2r & PC software (UNIVERSAL SOLUTIONS)
Procedure
Firstly, a sterile coverslip was placed in the bottom of the culture dish (forceps may be helpful) where cells stuck from the very beginning. Macrophages were then carefully seeded in 1 ml of supplemented Locke's solution at a density of 300,000- 500,000 cells per 35 mm culture dish. Since they are non-dividing cells they were used in the following two or three days after seeded (likely, after this lapse time, macrophages will be completely adhered to coverslips). Extensive washing with 500 μl PBS 1X (three-four replicates) was used to ensure that cells were very adherent and alive. The percentage of apoptotic/necrotic cells after 3 days in culture was below 5% (determined by using the common Trypan Blue staining, see Notes).
Note: Cell attaching to the coverslip is a critical step in order to perform the microfluorimetric analysis. If your plastic or glass interferes with the adherence of the macrophages, allow the support to dry with FCS (fetal calf serum) under the cabin to improve adherence. Avoid a high density cell culture. A 50-75% subconfluent culture with some cells interacting is desirable.
Incubate macrophages in 1 ml supplemented Locke's solution loaded with 5-7 μM Fura-2-AM (dissolved in DMSO, following the datasheet instructions) for 45 min approximately at 37 °C. Incubation does NOT involve either shaking or agitation, just a short and slightly balancing.
Wash thoroughly the culture monolayer with fresh 500 μl Locke's solution (serum also contains esterases that may degrade Fura-2-AM) and place the coverslip in a small superfusion chamber (34 μl volume). Junctions between the coverslip and the chamber are sealed with vacuum grease. Figure 1 depicts the superfusion chamber apparatus.
Note: Locke’s media or products perfused to the chamber are regulated using a valve system that works by gravity. The perfusion solutions are maintained in a water bath at 37 °C and the flow rate is kept constant at 1.5 ml/min. The vacuum pump aspires continuously the perfusion media after arriving to the chamber to prevent the accumulation of the hydrolysis products.
Figure 2 shows the experimental setup described here.
Figure 1. The superfusion chamber system. In the upper side of the figure, we can appreciate the superfusion chamber and the microscope stage. Immediately below these two pictures, both pieces are already assembled. The coverslip should be placed in the circular hollow remaining in the center.
Figure 2. Experimental equipment for Fura-2-AM registers. The temperature and the valve controllers, the fluorescence microscope (including its optical filter changer) and the necessary equipment to record the fluorescence images (the camera and its controller) are depicted above.
Images of both control and treated cells are visualized using the Plan Fluor x20/0.5 objective of the microscope. In this regard, Fura-2-AM has entered the cell and intracellular esterases have hydrolyzed the compound providing free Fura-2 that senses Ca2+ with a high affinity. Fluorescence emission occurs in a broad range around 430 nm when the cells are excited at 340 nm.
Note: In our study (Traves et al., 2013), macrophages were preincubated with different prostanoids for at least 10 min and then stimulated for 30 s with a variety of purinergic receptor agonists at near-maximal effective concentrations: 100 μM ATP, 100 μM UTP, 10 μM UDP, 10 μM 2MeSADP, 1,000 μM α,β-meATP or 300 μM BzATP.
Excite cells for 300 ms at 340/380 nm (< 5 ms wavelength change) and select the emitted light using the dichroic mirror (430 nm) and a 510-nm band-pass filter.
Note: The selection of these wavelengths matches with the maximum fluorescence registers for Fura-2 calcium-saturated solutions (340 nm) and calcium-free Fura-2 solutions (380 nm).
Fluorescence images are acquired with the camera every 1.5 seconds and controlled by the software. Sampling frequency is 2 Hz.
Data analysis
Images are processed by averaging signals from small elliptical regions within individual cells (Figure 3). The possibility exists to define specific areas of changes in the fluorescence emission (cell contact interactions, protrusions in the cytoplasm –cell polarization- or simply cells with different morphologies –round vs. shaped macrophages, depending on the treatments-).
Note: Background signals are subtracted from each wavelength.
Figure 3. Metafluor screenshot of cells. ROIs (cells in which a shift in the fluorescence emission is recorded as a function of time) and background (normally areas without cells or non-responsive cells during the period of observation) are marked with a dot in the image. The scale bar on the left side refers to the time variable (in seconds).
The F340/F380 ratio is calculated on the basis of the initial peak magnitude that represents the initial transient components (Figure 4). The F340/F380 ratio is converted into a known calcium concentration using the Grynkiewicz equation:
[Ca2+] = Kd * (R – Rmin) / (Rmax – R) * F380max/F380min (Grynkiewicz et al., 1985)
Where:
Kd is the dissociation constant (depends on the indicator, but also on pH, ionic strength, cell line, etc.).
R is the observed fluorescence ratio at both wavelengths (F340/F380).
Rmin is the minimum ratio value (in absence of Ca2+).
Rmax is the maximum ratio value (when Fura-2 is saturated by Ca2+).
F380max/F380min is a scaling factor (fluorescence intensity at 380 nm excitation in the absence of Ca2+ and at Ca2+ saturation).
Note: A calibration curve is required to calculate the Kd value. The F380max and F380min values are obtained at the end of each analysis in the same experimental conditions (Fura-2-AM concentration, exposition time…). It is based in two calibration points, a maximum and a minimum corresponding, respectively, to a saturated calcium solution (2.5 mM CaCl2) and a calcium-free solution (containing 10 μM EGTA). Alternatively, inhibition of the membrane reticulum Ca2+ pump with 200 nM thapsigargin or 500 nM ter-buthylbenzohydroquinone allow a saturation of the cytoplasmic Ca2+ concentration. Treatment of cells maintained in extracellular medium lacking or containing Ca2+ with a low dose of ionomycin (ca. 1 μM) to improve the entrance of the Ca2+ into the cell.
Figure 4. Fluorescence measured at 340/380 nm. A. Fluorescence intensity at both registered excitation wavelengths. B. Registers obtained over the time are divided to determine the F340/F380 ratio.
Notes
Trypan Blue staining can be used to discriminate between viable and non-viable cells. The protocol follows these typical steps:
Dilute the cell sample (1:10) to a total volume of 20 μl in a 0.4% Trypan Blue dye solution (should be sterile filtered before using).
While non-viable cells will be blue, viable cells will be unstained.
Carefully and continuously fill the hemocytometer chamber with 10 μl of the solution each chamber (all hemocytometers consist of two chambers; each is divided into nine 1mm2 squares).
Count cells under the microscope in four 1 x 1 mm squares of one chamber and determine the average number of cells per square. If the cell density is higher than 200 cells/square, you should dilute your cell suspension.
Total number of particles per ml in the cell sample can be calculated as follows: mean number of cells x 1/dilution factor x 104 cells/ml.
Recipes
Locke's Solution composition
140 mM NaCl
4.7 mM KCl
2.5 mM CaCl2
1.2 mM KH2PO4
1.2 mM MgSO4
5.5 mM glucose
10 mM HEPES, pH 7.4
Acknowledgments
This work was supported by grants CP11/00080 from ISCIII, BFU2011-024760 from MICINN and FIS-RECAVA RD12/0042/0019. RECAVA and Ciberehd networks are funded by the Carlos III Health Institute. A summary of the procedure was described in Traves et al. (2013).
References
Traves, P. G., Pimentel-Santillana, M., Carrasquero, L. M., Perez-Sen, R., Delicado, E. G., Luque, A., Izquierdo, M., Martin-Sanz, P., Miras-Portugal, M. T. and Bosca, L. (2013). Selective impairment of P2Y signaling by prostaglandin E2 in macrophages: implications for Ca2+-dependent responses. J Immunol 190(8): 4226-4235.
Grynkiewicz, G., Poenie, M. and Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6): 3440-3450.
Traves, P. G., Pimentel-Santillana, M., Carrasquero, L. M., Perez-Sen, R., Delicado, E. G., Luque, A., Izquierdo, M., Martin-Sanz, P., Miras-Portugal, M. T. and Bosca, L. (2013). Selective impairment of P2Y signaling by prostaglandin E2 in macrophages: implications for Ca2+-dependent responses. J Immunol 190(8): 4226-4235.
Tsien, R. Y., Rink, T. J. and Poenie, M. (1985). Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6(1-2): 145-157.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
González-Ramos, S., Carrasquero, L. M. G., Delicado, E. G., Miras-Portugal, M. T., Fernández-Velasco, M. and Boscá, L. (2013). Determination of the Intracellular Calcium Concentration in Peritoneal Macrophages Using Microfluorimetry. Bio-protocol 3(23): e988. DOI: 10.21769/BioProtoc.988.
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Category
Immunology > Immune cell function > Macrophage
Biochemistry > Other compound > Ion > Calcium
Cell Biology > Cell imaging > Microfluidics
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989 | https://bio-protocol.org/en/bpdetail?id=989&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Autoradiographic 3H-Gaboxadol Receptor Binding Protocol
LL Lynne Ling
Donald Caspary
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.989 Views: 7313
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Jan 2013
Abstract
Gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, THIP), a GABAA receptor δ-subunit specific agonist, when present at low (μM) concentrations, preferentially binds and activates extrasynaptic (non-γ2, δ-subunit-containing) GABAARs (Storustovu and Ebert, 2006; Richardson et al., 2011, 2013).
In this prototype saturation binding experiment, a series of concentrations of [3H]gaboxadol (5, 10, 25, 50, 75, 100, 250 and 400 nM) will be used. GABA at 200 μM will be added into binding mixtures as a cold displacer for [3H]gaboxadol. Slide mailers are used and each requires 7 ml binding mixture. Pre-, post-washing and binding buffer is 50 mM Tris-Citrate (pH 7.1). The detailed procedure is outlined below.
Materials and Reagents
Ice
[3H]gaboxadol (Gift from Merck & Co.)
GABA (Sigma-Aldrich, catalog number: A2129 )
Incubation buffer: 50 mM Tris-Citrate buffer (pH 7.1)
0.1 M PBS (Phosphate buffer saline, pH 7.4) Binding mixture (see Recipes)
Equipment
Slide mailer (Fisher Scientific, model: HS15986 )
Labeled foil
Cryostat (Leica Microsystems, model: CM1850 )
-20 ℃ freezer
Scintillation counter
Ruler
Diamond scribe
Double-sided tape
Phosphor transcreens
Cyclone Phosphor Imager (PerkinElmer)
Software
OptiQuant Version 4.0 (PerkinElmer)
GraphPad Radioactive Calculator (GraphPad Software)
Procedure
Animal brains will be removed after a quick decapitation and dipped in the PBS slush (0.1 M, pH 7.4) to remove surface blood. Once the brain tissue is frozen in dry ice, it can be wrapped in labeled foil and stored at -80 °C.
Sixteen micron thickness frozen sections will be cut in a cryostat, collected on slides and stored at -20 °C freezer for less than 48 h.
Slides will be brought to room temperature on the experiment day; DO NOT open the box until slides have equilibrated to room temperature (about 20 min at room temperature) in order to avoid excessive moisture on tissue sections. Afterwards, slides will be selected to be used for the experiment.
Wash with 50 mM Tris-Citrate buffer (pH 7.1) at 4 °C for 5 min three times.
Let slides dry at room temperature (leave slides for 45 min to 1 h or longer).
While waiting for the slides to dry
Make “Shoot for” solutions:
Theoretical “Shoot for”: “Shoot for” is the stock solution used to make a series of dilutions for receptor saturation analysis. The range of [3H]gaboxadol concentrations will be determined based on the literature and previous experiments. The highest concentration will then be used to calculate a theoretical “Shoot for”. For example, if the highest concentration used in the experiment is 400 nM of [3H]gaboxadol, the total volume added to the slide mailer is 7 ml. The volume of the 10X stock displacer solution to be added to the mailer is 0.7 ml; therefore, 6.3 ml of X concentration of the “Shoot for” will be needed. Thus, the “Shoot for” will be calculated as:
X = (400 nM*7 ml)/6.3 ml, and X = 444.44 nM.
Volume (V) of the original [3H]gaboxadol required to make the theoretical “Shoot for”:
V (μl) = Vt * X * SA
SA is the radioactive ligand’s specific activity (Ci/mmol) provided by the manufacturer. In addition, the specific activity for the same ligand may vary between production batches;
X is the theoretical concentration of the “Shoot for” (nM);
Vt is the total volume of “Shoot for” needed to make the series dilution (L). Therefore, Vt = V1+V2+……+VHighest
For each concentration, there will be a “total” mailer (measuring total binding, displacer omitted) and a “blank” mailer (measuring non-specific binding, displacer added). For example, a series of concentrations of 5, 10, 25, 50, 75, 100, 250 and 400 nM [3H]gaboxadol are used and the total volume of the “Shoot for”:
Vt (L) = [(5 nM*7 ml/X)+(10 nM*7 ml/X)… +(400 nM*7 ml/X)]*2
1000
The “Shoot for” solution will be made by adding V (μl) of the original [3H]gaboxadol to Vt-V amount of the incubation buffer.
Count “Shoot for” using Scintillation counter by aliquoting 10 μl of “shoot for” into 5 ml of scintillation fluid. Based on the counts, calculate the actual concentration of the “Shoot for” using GraphPad Radioactive Calculator. If the measured value is close to the theoretical concentration, continue to the next step. From this point forward, the actual concentration of the stock solution will be used for the dilution calculations.
Pipetting required amount of 50 mM Tris-Citrate buffer to tubes labeled with Total or Blank of 5, 10, 25, 50, 75, 100, 250 and 400 nM of [3H]gaboxadol, add appropriate volume of the stock solution to make to the designated concentrations.
Add 0.7 ml of 200 μM GABA into tubes labeled Blank.
Count the counts of the mixture in 16 tubes. The actual concentration of [3H]gaboxadol in each tube will be calculated based on these counts.
Transfer mixtures from 16 tubes into the correctly labeled slide mailer.
Drop slides into their designated mailers. Shake well, before leaving mailers in the refrigerator for 1 h at 4 °C.
After incubation, slides will be washed twice in cold Tris-Citrate buffer for 10+10 sec, followed by cold dH2O for 10 sec.
Let slides dry for 2-4 h or overnight.
Break the slides: re-label the slides next to the sections, line up sections with a ruler and cut the slides using a diamond scribe. The slide end (with one section on it) will be taped using double-sided tape after being broken.
Put the slides onto the phosphor transcreens; transcreen exposure time is about 48 h.
Binding images are obtained by scanning the exposed phosphor transcreens using a Cyclone Phosphor Imager.
Figure 1. An autoradiographic image of [3H]gaboxadol on GABAA receptor binding in a four month old Fischer Brown Norway rat brain tissue (16 μm thickness). Highest binding area (circled) is the medial geniculate body, while hippocampus and cortex show moderate binding.
Recipes
Binding mixture
Binding mixture consists of 50 mM Tris-Citrate buffer and [3H]gaboxadol with/without 200 μM GABA
Acknowledgments
This protocol is adapted from Richardson et al. (2011) and Richardson et al. (2013). Work supported by NIH DC000151 to DMC.
References
Richardson, B. D., Ling, L. L., Uteshev, V. V. and Caspary, D. M. (2013). Reduced GABA(A) receptor-mediated tonic inhibition in aged rat auditory thalamus. J Neurosci 33(3): 1218-1227a.
Richardson, B. D., Ling, L. L., Uteshev, V. V. and Caspary, D. M. (2011). Extrasynaptic GABA(A) receptors and tonic inhibition in rat auditory thalamus. PLoS One 6(1): e16508.
Storustovu, S. I. and Ebert, B. (2006). Pharmacological characterization of agonists at delta-containing GABAA receptors: Functional selectivity for extrasynaptic receptors is dependent on the absence of gamma2. J Pharmacol Exp Ther 316(3): 1351-1359.
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Neuroscience > Cellular mechanisms > Receptor-ligand binding
Biochemistry > Protein > Interaction
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99 | https://bio-protocol.org/en/bpdetail?id=99&type=0 | # Bio-Protocol Content
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Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell
YC Yanling Chen
Published: Vol 2, Iss 4, Feb 20, 2012
DOI: 10.21769/BioProtoc.99 Views: 71656
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Abstract
Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, usually a tissue-culture-treated microporous membrane, which is positioned between two compartments that mimic two different sets of microenvironments for cell survival/growth. Cells on one side of the membrane, when sensing chemoattractants placed on the other side of the compartment that diffuses through the membrane, can migrate through the pores in the membrane towards the source of the chemoattractants. Cells that migrate across the membrane can be quantified by fixing and counting. Human breast epithelial adenocarcinoma MD-231 cells grow relatively fast and are metastatic. The MB-231 cell line is used here to describe the procedures of an in vitro cell migration assay using the transwell apparatus.
Materials and Reagents
Human MDA-MB-231 cell (ATCC, catalog number: HTB-26 ™)
Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
Trypsin inhibitor (soybean) (Life Technologies, Invitrogen™, catalog number: 17075-029 )
Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
Collagen I (Sigma-Aldrich, catalog number: C7661 ) or Fibronectin (BD Biosciences, catalog number: 354008 )
Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
Ethanol (Sigma-Aldrich, catalog number: 459836 )
Crystal violet (Sigma-Aldrich, catalog number: C3886 )
TCC-formulated Leibovitz's L-15 Medium (ATCC, catalog number: 30-2008 ™)
Equipment
Corning® Transwell® polycarbonate membrane inserts (Sigma-Aldrich, catalog number: CLS3421 ) or Millicell Cell Culture Inserts (EMD Millipore, catalog number: PI8P01250 )
Cotton swabs
Cell culture incubator: 37 °C and 5% CO2
Procedure
Carry MB-231 cells in DMEM with 10% FBS (use L-15 medium if needed).
Wash cells twice with 1x PBS and trypsinize.
Add 0.5 mg/ml Trypsin inhibitor in PBS to inactivate an equal volume of Trypsin. Aspirate cells by pipetting up and down gently (Note: It is important to break down into individual cells as much as possible).
Gently spin down the cells. Wash cells two times with DMEM containing 0.5% FBS to remove trace amounts of trypsin and inhitibor. Resuspend the cells in DMEM with 0.5% FBS and count.
Prepare the transwell compartments, 24-well format, with 8 μm pore size insert:
To the lower compartment, add 2.6 ml of DMEM with 0.5% FBS containing 40 μg ml-1 Collagen I.
Add the transwell insert to the well by merging the bottom of the insert into the medium in the lower compartment.
Note: Ensure that no air bubbles are trapped between the insert membrane and the medium.
To the upper compartment, gently add 1 x 105 cells from step 4.
Incubate the cells in the transwell plate at 37 °C and 5% CO2 for 2.5 h. This allows cells to migrate toward the underside of the insert filter.
After 2.5 h, carefully take the insert out. Cells that do not migrate through the pores and therefore remain on the upper side of the filter membrane need to be gently removed with a cotton swab. Gently wipe the upper side of the filter membrane with a cotton swab to remove the cell debris. We recommend that use each clean cotton swab for one wipe only, in one direction and do not swipe in back-and-forth movement. The cotton swab can be slightly moisturized with ddH2O as needed but be sure to remove any excess water. Several wipes may be needed to completely remove any cell debris on the membrane.
Fix the cells on the lower side of the insert filter quickly with 5% glutaraldehyde for 10 min.
Next, stain cells on the lower side of the insert filter with 1% crystal violet in 2% ethanol for 20 min.
Remove excess crystal violet by quickly merging the insert in ddH2O for 3 to 4 sec. Drain excess water from the side of the insert using a cotton swab. Dry the insert membrane.
Count the number of cells on the lower side of the filter under a microscope. Randomly choose different views and take average counting.
The same experimental procedure should be performed for control groups without chemoattractants. Each migration condition should be tested with replicates.
Acknowledgments
This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Katoh et al. (2006) and Nakamizo et al. (2005). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee [see Chen et al. (2009)].
References
Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
Katoh, H., Hiramoto, K. and Negishi, M. (2006). Activation of Rac1 by RhoG regulates cell migration. J Cell Sci 119(Pt 1): 56-65.
Nakamizo, A., Marini, F., Amano, T., Khan, A., Studeny, M., Gumin, J., Chen, J., Hentschel, S., Vecil, G., Dembinski, J., Andreeff, M. and Lang, F. F. (2005). Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 65(8): 3307-3318.
Article Information
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© 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Chen, Y. (2012). Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell. Bio-protocol 2(4): e99. DOI: 10.21769/BioProtoc.99.
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Category
Cancer Biology > General technique > Cell biology assays > Cell migration
Cancer Biology > Invasion & metastasis > Drug discovery and analysis > Cell migration
Cell Biology > Cell movement > Cell migration
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990 | https://bio-protocol.org/en/bpdetail?id=990&type=0 | # Bio-Protocol Content
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Peer-reviewed
Measurement of Potassium Content in Arabidopsis
Mitsuru Abo
YO Yuriko Osakabe
KY Kazuko Y-Shinozaki
EY Etsuro Yoshimura
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.990 Views: 9846
Reviewed by: Ru Zhang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Plant Cell Feb 2013
Abstract
Potassium is an essential element in plant growth and has an important role in regulating cell water potential and turgor in osmotic regulation. Potassium content in plants is high compared to trace elements, however, it is difficult to measure a relatively small change of potassium content in the large total. Here, we describe a procedure for measuring potassium in Arabidopsis that is easy to handle in preparative scale and avoids contamination.
Materials and Reagents
Arabidopsis seedlings
Milli-Q water
Nitric acid (analytical grade for heavy metals) (Wako Pure Chemical Industries, catalog number: 140-04016 )
Potassium standard solution (1,000 ppm) (Wako Pure Chemical Industries, catalog number: 165-17471 )
Contaminon L (Wako Pure Chemical Industries, catalog number: 035-09311 )
Murashige and Skoog salt (Wako Pure Chemical Industries, catalog number: 392-00591 )
2-(N-morpholino)ethanesulfonic acid (MES, Wako Pure Chemical Industries, catalog number: 345-01625 )
Gamborg’s Vitamin Solution (Sigma-aldrich, catalog number: G1019 )
Difco Bacto agar (Becton, Dickinson and Company, catalog number: 214010 )
Germination media (GM)-agar (see Recipes)
Equipment
Plates
Centrifugation tubes
A paper or aluminum foil
Atomic absorption spectrometer (AAS) or Inductively-coupled plasma optical emission spectrometer (ICP-OES)
Stainless-steel vessel (HU-25, o.d. = 55 mm x 104 mm) (SAN-AI science)
Polytetrafluoroethylene (PTFE) vessel (o.d. = 28 mm x 40 mm) (SAN-AI science)
Tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA) cup (o.d. = 22 mm x 32 mm, 7 ml) (Sanplatec, catalog number: 0225R )
Volumetric flask (100 ml)
Ceramic scissors and tweezers (AS ONE Corporation, catalog number: 8-203-23 , 7-166-11 )
Procedure
Arabidopsis seedlings are germinated for 10 days on GM agar and then cultivated hydroponically with milliQ H2O in plates for 5 days.
Plants are dissected into shoots and roots by cutting the bottom of the shoots with ceramic scissors and tweezers.
Samples are dried in disposable centrifugation tubes (50 ml) at 70 °C for 12 h. A paper or aluminum foil is placed over the samples during the drying process.
Dried samples (between 15 and 20 plants, corresponding to between 5 and 10 mg as dry weight) are placed into PFA cups with ceramic tweezers and weighed.
The PFA cups are placed in PTFE vessels, and nitric acid (1 ml) is added to the PTFE vessels outside the PFA cups. To avoid contamination of impurities in nitric acid, samples are decomposed by nitric acid vapor (not nitric acid liquid).
The PTFE vessels are set in stainless-steel vessels (HU-25) and heated at 150 °C for 10 h.
The decomposed samples in the PFA cups are diluted with 0.1 M nitric acid and poured into a volumetric flask (100 ml). The sample solution is filled up with 0.1 M nitric acid and subjected to analysis.
Potassium content in plants is measured with an atomic absorption spectrometer in emission mode. Standard solutions at concentrations of 0, 1, 2, 4, 8 ppm are prepared from a 1,000 ppm reference solution diluted with 0.1 M nitric acid (It also can be measured by ICP-OES).
Notes
To avoid potassium contamination from hands, use rubber gloves during sample treatments.
Take care when handling dried samples, which may be attached to vessel walls or scattered due to static electricity.
Dried samples are very hygroscopic. It is necessary to weigh them quickly for an accurate content calculation.
Contaminon L is used for heavy metal cleaning. Glass vessels are immersed in Contaminon L overnight and rinsed off with MilliQ water before use.
Recipes
GM (germination media)-agar
1x Murashige and Skoog salt
3% sucrose
1x Gamborg’s Vitamin solution
0.05% 2-(N-morpholino)ethanesulfonic acid (MES)
0.8% Difco Bacto agar
Adjusted to pH 5.7 with 1 M KOH
Acknowledgments
This protocol is based on the procedure described by Kojima and Iida (1986).
References
KOJIMA, I. and IIDA, C. (1986). Phase digestion of botanical samples polytetrafluoroethylene bomb. Anal Sci 2: 567.
Osakabe, Y., Arinaga, N., Umezawa, T., Katsura, S., Nagamachi, K., Tanaka, H., Ohiraki, H., Yamada, K., Seo, S. U., Abo, M., Yoshimura, E., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2013). Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis. Plant Cell 25(2): 609-624.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Abo, M., Osakabe, Y., Y-Shinozaki, K. and Yoshimura, E. (2013). Measurement of Potassium Content in Arabidopsis. Bio-protocol 3(23): e990. DOI: 10.21769/BioProtoc.990.
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Category
Plant Science > Plant physiology > Ion analysis
Biochemistry > Other compound > Ion > Potassium
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991 | https://bio-protocol.org/en/bpdetail?id=991&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood
Qingfeng Chen
JC Jianzhu Chen
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.991 Views: 14235
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Original Research Article:
The authors used this protocol in Stem Cells Jun 2013
Abstract
CD34 is a glycosylated cell surface protein and represents a well-known marker for primitive progenitor cells in various organs, especially cord blood, bone marrow and fetal liver. CD34+ progenitor cells are suitable for a series of studies, e.g. cell differentiation, transplantation as well as construction of humanized mouse models. Here, we describe a method to isolate CD34+ cells from the human cord blood and fetal liver.
Keywords: Hematopoietic stem cells Cord Blood Fetal liver Magnetic purification Hepatic progenitor cells
Materials and Reagents
Collagenase, Type IV (Life Technologies, catalog number: 17104019 )
Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
ACK Lysing Buffer (Life Technologies, catalog number: A1049201 )
RoboSepTM Buffer (STEMCELL Technologies, catalog number: 20104 )
Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
EasySepTM Human Cord Blood CD34 Positive Selection Kit (STEMCELL Technologies, catalog number: 18096 )
StemSpanTM SFEM (STEMCELL Technologies, catalog number: 0 9650 )
DMSO Hybri-Max Sterilefilt (Sigma-Aldrich, catalog number: D2650 )
Hyclone Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 30070.03 )
Liquid nitrogen
1 mg/ml Collagenase IV (see Recipes)
Freezing medium (see Recipes)
PBS/EDTA (2 mM) (see Recipes)
Equipment
Syringe Filter (0.20 μm Blue Rim) (Minisart®, catalog number: 16534-K )
Centrifuge Tube (50 ml Blue Cap) (BD Biosciences, Falcon®, catalog number: 35 2070 )
T75 cell culture flask
Petri dish (90 x 15 mm) (Thermo Fisher Scientific, catalog number: BSN 101VR20 )
Cell scrapers (L29 cm Blade 2 cm) (SPL Life Sciences Co., catalog number: 90030 )
Shaker Incubator (New Brunswick Scientific, model: Innova 4000 )
Sterile wire mesh (100 μm) (cut larger than the size of a 90 x 15 mm Petri dish)
Syringe (10 cc Luer Lok) (BD, catalog number: DS-BD-15026C )
Tabletop Centrifuge Legend RT (Sorvall)
FalconTM Polystyrene Round-Bottom Tubes (14 ml) (BD, catalog number: 352057 )
Big EasySep® Magnet (STEMCELL Technologies, catalog number: 18001 )
Hematocytometer (TOMY DIGITAL BIOLOGY, catalog number: DHC-N01 )
Cryovial
Cryo tube (free standing 2 ml) (Corning Incorporated, catalog number: 430488 )
Mr. Frosty Freezing Container (Cyro 1DEGC) (Thermo Fisher Scientific, catalog number: PLW- FS-00033 )
Leucosep tube
37 °C incubator
Centrifuge
Procedure
Part I: Isolation of CD34+ cells from fetal liver
Processing fetal liver
Weigh out Collagenase IV for a final concentration of 1 mg/ml Collagenase IV with DMEM.
Dissolve Collagenase IV with 10 ml of DMEM and filter the suspension using a 0.2 μm filter. Divide the suspension equally into 2 x 50 ml Falcon tubes.
Place the fetal liver in a petri dish filled with 20 ml DMEM.
Cut liver into small pieces using cell scrapers at room temperature. Mix the suspension well and divide the volume evenly into the prepared Falcon tubes.
Add more DMEM to the petri dish to wash down excess tissue and transfer into the same prepared Falcon tube.
Bring the tissue suspension to a total volume of 40 ml per Falcon tube with DMEM.
Incubate 37 °C for 30 min with shaking 200 rpm.
Place a sterile 100 μm wire mesh in the petri dish.
Filter tissue suspension through the 100 μm wire mesh. Grind non-filtered tissue particles with the end of a 10 ml plunger against the mesh. Ensure that there are no remaining clumps.
Transfer filtered medium into a fresh 50 ml Falcon tube.
Spin at 400 x g for 5 min with a tabletop centrifuge.
Remove supernatant.
Lyse red blood cells
Add 10 ml of ACK lysing buffer to the pellet and resuspend well.
Incubate for 3 min at room temperature.
Neutralise the buffer with 10 ml of DMEM. Resuspend.
Spin at 400 x g for 5 min at room temperature.
Remove supernatant.
CD34 selection
Resuspend cell pellet with 4-16 ml of Robosep buffer depending on the gestation age of the fetal liver and do a cell count using hematocytometer.
Place cells in a 14 ml polystyrene tube (up to 4 ml per tube) and prepare cells to a concentration of 2 x 108 cells/ml.
Add EasySepTM Positive Selection Cocktail at 120 μl/ml cells (e.g. for 5 ml of cells, add 600 μl of cocktail). Mix well and incubate at room temperature for 15 min.
Pipette EasySepTM Magnetic Particles vigorously more than 5 times. Do not vortex.
Add the particles at 50 μl/ml cells (e.g. for 5 ml of cells, add 250 μl of nanoparticles). Mix well and incubate at room temperature for 10 min.
Bring the cell suspension to a total volume of 10 ml with Robosep buffer. Gently resuspend cells before placing the tube (without cap) into the magnet. Set aside for 5 min.
Pick up the EasySep Magnet, and in one continuous motion invert the magnet and tube, pouring off the supernatant fraction. The magnetically labelled cells will remain inside the tube, held by the magnetic field of the EasySep Magnet. Leave the magnet and tube in inverted position for 2-3 seconds, then return to upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube.
Remove the tube from the magnet and add 10 ml of Robosep buffer. Gently resuspend the cells and place the tube back into the magnet. Set aside for 5 min.
Repeat step 24-25, and do a total of 4 washes.
After the last wash, resuspend cells and combine cells from different tubes with 5 ml of Robosep buffer.
Perform a cell count using hematocytometer.
Spin cells down at 400 x g for 5 min.
Cryopreservation
Prepare freezing medium (1.2 ml per 5 million cells).
Resuspend cells with freezing medium and aliquot 1.2 ml into each cryovial.
Place cryovials in Mr Frosty and leave it overnight.
Transfer cryovials to liquid nitrogen the next day.
Part II: Isolation of cord blood CD34+ cells
Pre-enrichment
Pour the cord blood sample into the T75 cell culture flask.
Add RosetteSep Cord Blood CD34 Pre-enrichment cocktail at 5 μl/ml of cord blood and gently pipette until thoroughly mixed.
Incubate at room temperature for 10 min.
Dilute the blood sample with equal volume (1:1) of PBS + 2 mM EDTA and mix gently.
Isolation of mononuclear cells
Add 15 ml of RT Ficoll-Plaque Plus into Leucosep tube.
Centrifuge at 1,000 x g for 30 seconds at RT to allow the Ficoll-Plaque Plus get into the bottom part of the filter in Leucosep tube.
Add 30 ml of diluted blood sample into the LeucoSep + Ficoll tubes.
Turn Off The Centrifuge Brake!!! And centrifuge at 1,000 x g for 20 min at RT.
Remove the plasma layer and collect the buffy coats layer into a new 50 ml falcon tube.
Top up 40 ml of PBS + 2 mM EDTA to wash the cells.
Turn On The Centrifuge Brake!!! And centrifuge for 15 min at 400 x g.
Discard the supernatant and resuspend the cell pellet with 10 ml of ACK lysis buffer.
Incubate at room temperature for 5 min for RBC to lyse.
Quench the sample with 40 ml PBS + 2 mM EDTA (1:4 dilution).
Centrifuge for 15 min at 400 x g.
Resuspend in 1 ml of Robosep buffer and perform cell counting using hematocytometer.
CD34 selection and cryopreservation
Repeat step 18 to 33 in Part one.
Recipes
1 mg/ml Collagenase IV
On average use a final volume of 80 ml for a 16-19 weeks old foetus
or 160 ml for a 20-24 weeks old foetus
Make fresh before use
Freezing medium
For every 5 million cells/cryovial, volume of 1.2 ml:
600 μl StemSpan
510 μl Heat induced-FBS
90 μl dimethylsulfoxide DMSO
Ensure mixture is homogenous before resuspending with cell pellet.
PBS/EDTA (2 mM)
Add 2 ml of 0.5 M EDTA stock to 500 ml 1x PBS (Filtered)
Acknowledgments
This protocol was developed and adapted from the previous publication Chen et al. (2013).
References
Chen, Q., Khoury, M., Limmon, G., Choolani, M., Chan, J. K. and Chen, J. (2013). Human Fetal Hepatic Progenitor Cells Are Distinct from, but Closely Related to, Hematopoietic Stem/Progenitor Cells. Stem Cells 31(6): 1160-1169.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Chen, Q. and Chen, J. (2013). Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood. Bio-protocol 3(23): e991. DOI: 10.21769/BioProtoc.991.
Download Citation in RIS Format
Category
Cell Biology > Cell isolation and culture > Cell isolation
Stem Cell > Adult stem cell > Hematopoietic stem cell
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992 | https://bio-protocol.org/en/bpdetail?id=992&type=0 | # Bio-Protocol Content
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Peer-reviewed
Serial Transfer of Human Hematopoietic and Hepatic Stem/progenitor Cells
Qingfeng Chen
JC Jianzhu Chen
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.992 Views: 14301
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Original Research Article:
The authors used this protocol in Stem Cells Jun 2013
Abstract
A range of assays have been developed to determine the stemness or stem cell activity of human stem cells. The key assays of stem cells are functional: they must show self-renewal and the ability to generate the appropriate tissue. The best assays available to study this property in putative human stem cells involve xeno-transplantation into immune-deficient mice. Demonstration of both long-term (2-3 months) multi-lineage reconstitution of human blood or liver in a murine host and the ability of the putative stem cells to mediate reconstitution of a secondary host upon re-isolation from the primary mouse are generally accepted as the gold standard for demonstrating the presence of human hematopoietic and hepatic stem cells. Here, we describe a method of reconstituting NOD-scid IL-2Rγ-/-(NSG) mice with CD34+ stem cells from human fetal liver and repurification of CD34+ cells for serial transplantation.
Keywords: Hematopoietic progenitor Hepatic progenitor Immune system Human Hepatocyte Chimera mouse
Materials and Reagents
NOD.Cg-Prkdcscid //2rgtm1Wjl/SzJ (The Jackson Laboratory, stock number: 00 5557 )
CD34+ fetal liver cells
StemSpanTM SFEM (STEMCELL Technologies, catalog number: 0 9650 )
ACK Lysing Buffer (Life Technologies, catalog number: A1049201 )
Liver perfusion medium (Life Technologies, catalog number: 17701-038 )
Liver digestion medium (Life Technologies, catalog number: 17703-034 )
RoboSepTM Buffer (STEMCELL Technologies, catalog number: 20104 )
Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
EasySepTM Human Cord Blood CD34 Positive Selection Kit (STEMCELL Technologies, catalog number: 18096 )
DMEM
Equipment
Biosafety cabinet
Petri dishes (100-mm2)
137 Cs gamma irradiator
Insulin syringe (29 G 1 cc) (BD Biosciences, catalog number: 320310 )
Syringe (5 ml) (BD, catalog number: 309646 )
Heating pad or warming lamp
Butterfly needle (BD, catalog number: 368659 )
Cell strainer (100 μm) (BD Biosciences, catalog number: 352360 )
Falcon tube(15 ml)
Procedure
Engraftment of primary recipient mouse
CD34+ fetal liver cells are purified based on the protocol “Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood” (Chen and Chen, 2013)
Monitor breeder pairs for the birth of new litters.
Note: Engraftment procedures should be performed on newborn pups 24 to 48 h post-natal.
Prepare CD34+ fetal liver cells suspend in StemSpan at 2.5 x 105 cells/50 μl/pup.
Note: Freshly prepared or previously frozen preparations may be used.
Place 24- to 48-h post-natal pups from a single litter into a 100 mm2 petri dish along with a small amount of bedding material from the breeder cage.
The petri dish is put into a 137 Cs gamma irradiator. Irradiate pups with 1 Gy whole body irradiation.
The petri dish is then brought back into biosafety cabinet. A second sterile petri dish is prepared with cotton nestlet from parent cage.
One pup is taken at a time from the irradiated dish and held firmly, yet with great care, by thumb and index finger of one hand. Tilt the pup back so that abdomen is exposed. The liver will be visible on the right flank of the pup (Figure 1). Disinfect area with alcohol pad.
Figure 1. Liver: site of injection
The other hand holds a 29 G 1 cc insulin syringe loaded with 50 μl of fetal liver cells.
The needle (perpendicular to body) will be inserted straight in, with bevel facing upwards, approximately 3 mm into the pup.
The cells are then carefully and slowly injected into the pup's liver.
Once injected, the needle is removed and gentle pressure is applied to the area. The injected pup is then placed in the second petri-dish.
Steps A7-11 are repeated until all the pups have been injected.
The pups are carefully placed back into their parents’ cage and covered with the cotton nestlet so they will smell familiar to parents.
The pups will be monitor everyday for seven days. Any pups exbihiting severe weight loss, dehyration, dyspnea should be euthanized immediately. From experience, the pups are mostly unaffected by the injection.
Cell repurification and reconstitution of secondary recipient mouse
8 to 10 weeks later, the primary mice are used for repurification of human hematopoietic stem cells and hepatic progenitor cells respectively.
For repurification of hematopoietic stem cells from femurs
Femurs are harvested and Remove as much muscle as possible around the femur bone.
Attach a 27 G needle to a 5 ml syringe filled with PBS.
Place the needle into the bone marrow (red middle of the bone), and flush out cells onto a 100 μm cell strainer in a 5 cm petri dish.
Mesh cells and transfer the pass-through to a 15 ml Falcon tube.
Pellet cells at 400 x g for 5 min.
Lyze red blood cells with ACK lysis buffer.
Re-purify CD34+ cells by magnetic selection with EasySepTM Human Cord Blood CD34 Positive Selection Kit.
For repurification of hepatic progenitor cells from livers
Cannulate portal vein (Figure 2) with a 27 G buffer fly needle.
Make incision in inferior vena cava (Figure 2).
Figure 2. Portal vein and inferior vena cava
Mouse livers were first perfused with pre-warmed liver perfusion medium at 0.7 ml/min for 10 min, then with pre-warmed liver digestion medium for 10 min.
Carefully transfer the digested liver to a petri dish and dis-associate liver cells into single cell suspensions with curved forceps.
The cell suspensions were washed with ice-cold DMEM at 50 x g for 5 min.
Assay cell viability by Trypan Blue dye.
CD34+ cells were re-purified from the cell suspensions with EasySepTM Human Cord Blood CD34 Positive Selection Kit.
Reconstitution of secondary recipients
CD34+ cells of desired numbers were injected into sublethally irradiated newborn NSG pups.
After 8 to 10 weeks, samples e.g. blood and livers are harvested for analysis.
Acknowledgments
This protocol was developed and adapted from the previous publication Chen et al (2013a) and (2013b).
References
Chen, Q., Khoury, M., Limmon, G., Choolani, M., Chan, J. K. and Chen, J. (2013a). Human fetal hepatic progenitor cells are distinct from, but closely related to, hematopoietic stem/progenitor cells. Stem Cells 31(6): 1160-1169.
Chen, Q. and Chen, J. (2013b). Isolation of CD34+ cells from human fetal lLiver and cord blood. Bio-protocol 3(23): e991.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Chen, Q. and Chen, J. (2013). Serial Transfer of Human Hematopoietic and Hepatic Stem/progenitor Cells. Bio-protocol 3(23): e992. DOI: 10.21769/BioProtoc.992.
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Category
Stem Cell > Adult stem cell > Maintenance and differentiation
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Great protocol. Thank you. Have you noticed any internal bleeding in these mice and how did you deal with it?
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993 | https://bio-protocol.org/en/bpdetail?id=993&type=0 | # Bio-Protocol Content
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Peer-reviewed
Longitudinal Bioluminescent Quantification of Three Dimensional Cell Growth
Michael K. Wendt
William P. Schiemann
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.993 Views: 8733
Reviewed by: Lin Fang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Oncogene Apr 2013
Abstract
The use of three-dimensional (3D) cell culture systems is widely accepted as representing a more physiologically relevant means to propagate mammary epithelial and breast cancer cells. However, 3D cultures systems are plagued by several experimental and technical limitations as compared to their traditional 2D counterparts. For instance, quantifying the growth of mammary epithelial or breast cancer organoids longitudinally is particularly troublesome using standard [3H]thymidine or MTT assay systems, or using computer-assisted area calculations. Likewise, the nature of the multicellular aggregates and organoids formed by breast cancer cells under 3D conditions precludes efficient recovery of the cells from 3D matrices, an event that is time consuming and leads to spurious results. The assay described here utilizes stable expression of firefly luciferase as means to quantify the longitudinal outgrowth of cells propagated within a 3D matrices. The major advantages of this technique include its high-throughput nature and ability to longitudinally track single wells over a defined period of time, thereby decreasing the costs associated with assay performance. Finally, this technique can be readily combined with drug treatments and/or genetic manipulations to assay their effects on the growth of 3D organoids.
Keywords: Proliferation Bioluminescence 3D-cultures In vitro outgrowth
Materials and Reagents
Murine 4T1 mammary carcinoma cells (ATCC, catalog number: CRL-2539 ) or any cell line of interest engineered to stably express firefly luciferase under the control of a constitutively-active promoter such cytomegalovirus.
Note: Several Luciferase encoding plasmids are commercially available and typically employ pcDNA3.1- or pBabe-based backbones to deliver firefly or renilla luciferases. In either scenario, antibiotic administration is used to select and maintain stable expression of luciferase in reporter cells.
Cultrex: Reduced growth factor (RGF) basement membrane extract (BME) (Trevigen, catalog number: 3433-005-01 )
Ice
Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, catalog number: 10313-021 )
Penicillin/Streptomycin (Pen/Strep) (Gibco®, catalog number: 15140 )
Fetal bovine serum (Sigma-Aldrich, catalog number: F1051 )
D-luciferin, Potassium Salt (15 mg/ml) in sterile H2O (Gold Bio, catalog number: LUCK-1G )
Equipment
2D culture dishes
White walled, clear bottom 96-well culture dishes (Corning, Costar®, catalog number: 3610 )
Luminometer capable of reading 96-well plate format (Promega GloMax-Multi Detection System or similar bioluminescent plate reader).
Hemocytometer or other means of cell counting
37 °C/5% CO2 cell incubator
Procedure
Cells are grown in DMEM supplemented with 10% FBS and Pen/Strep (full growth media).
Cells are harvested from actively proliferating, sub-confluent 2D culture dishes.
Cells are trypsinized, washed in excess full-growth media, and pelleted by gentle centrifugation. Afterward, the resulting cell pellets are resuspended and allowed to recover in full growth media for 2 h.
During this time, coat the 96-well dish with 50 μl of 100% Cultrex per well and allow to gel at 37 °C.
Note: Cultrex must be maintained on ice at all times to prevent solidification of the matrices that transpires as the gel warms to >4 °C.
Count the cells using the hemocytometer and dilute them to 1,000 cells in 150 μl of full growth media, which is supplemented with 4% Cultrex. Thoroughly mix cells and media/4% Cultrex solution and subsequently plate the cells on top of the solidified Cultrex cushions.
Note: The 4% Cultrex solutions do not solidify, and as such, cells contained within these mixtures will readily attach to solidified Cultrex cushions, thereby permitting top layer media changes and/or replacements throughout the experiment.
Prior to plating the cells, other compounds such as growth factors or chemical inhibitors may be mixed with the cells and 4% Cultrex solutions. Each experimental condition should be plated in triplicate.
Note: All test compounds must be screened initially by short-term exposure to verify that these agents do not directly impact the expression of CMV-driven luciferase and/or the activity of luciferase:luciferin reactions.
Two hours after plating the cells, obtain initial time zero (T0) luminescence readings by adding 2 μl of D-luciferin (15 mg/ml) and gently tap the side of the plate to mix.
Note: Culture lid is open while in the plate reader, and as such, it is imperative that the plate reader remain clean and well-sanitized, and potentially be located in a laminar flow hood to prevent unwanted cell contamination. Culture medium does not need to be replaced at this time.
Place culture in a 37 °C incubator with 5% CO2.
Four days after plating, multicellular organoids will have begun to form. Obtain a second luminescence reading as described in step 7. Afterward, change and refresh all media and experimental test components, being extremely careful not to disrupt the cells and solidified Cultrex cushion.
Note: At this point, therapeutic protocols can be initiated to monitor the anticancer activities of various chemotherapies against established organoids.
For all experimental conditions, repeat steps 7-9 at days 8 and 11 post-plating.
Note: These time points may vary depending dramatically based on the relative growth characteristics of the cell line under study. As such, longitudinal luciferase readings, dosing regiments, and cell plating densities need to be determined empirically for each cell line to be studied to maximize signal-to-noise ratios.
Use Excel to normalize each luminescence value to the initial T0 value derived after plating cells.
Acknowledgments
We thank the members of the Schiemann laboratory for their helpful comments and suggestions. Bioluminescent 3D-organotypic longitudinal growth assays were adapted from Wendt et al. (2011) and Wendt et al. (2013). Research support was provided in part by the National Institutes of Health to M.K.W. (CA166140), and to W.P.S. (CA129359 and CA177069).
References
Wendt, M. K., Schiemann, B. J., Parvani, J. G., Lee, Y. H., Kang, Y. and Schiemann, W. P. (2013). TGF-beta stimulates Pyk2 expression as part of an epithelial-mesenchymal transition program required for metastatic outgrowth of breast cancer. Oncogene 32(16): 2005-2015.
Wendt, M. K., Taylor, M. A., Schiemann, B. J. and Schiemann, W. P. (2011). Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer. Mol Biol Cell 22(14): 2423-2435.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Wendt, M. K. and Schiemann, W. P. (2013). Longitudinal Bioluminescent Quantification of Three Dimensional Cell Growth. Bio-protocol 3(23): e993. DOI: 10.21769/BioProtoc.993.
Download Citation in RIS Format
Category
Cancer Biology > General technique > Cell biology assays > Proliferation analysis
Cell Biology > Cell isolation and culture > 3D cell culture
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994 | https://bio-protocol.org/en/bpdetail?id=994&type=0 | # Bio-Protocol Content
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Peer-reviewed
Escherichia coli Outer Membrane Vesicle Immunization Protocol and Induction of Bacterial Sepsis
Oh Youn Kim
BH Bok Sil Hong
KP Kyong-Su Park
YY Yae Jin Yoon
SC Seng Jin Choi
WL Won Hee Lee
TR Tae-Young Roh
YK Yoon-Keun Kim
Yong Song Gho
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.994 Views: 12570
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Original Research Article:
The authors used this protocol in The Journal of Immunology Apr 2013
Abstract
Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. OMVs, which resemble the outer membrane and periplasm in composition, are proinflammatory and immunogenic facsimiles, and therefore could activate both innate and adaptive immunity. Here, we describe the OMVs immunization protocol and bacteria challenge protocol to induce bacterial sepsis in mice.
Keywords: Outer membrane vesicles Sepsis Animal model Bacterial extracellular vesicles Immunization
Materials and Reagents
Five-week-old male C57BL/6 or BALB/c mice or a variety of transgenic/knockout mice, body weight 18-20 g
Phosphate buffered saline (PBS) (Gibco®, catalog number: 70013-032 )
Escherichia coli (E. coli) derived OMVs
E. coli (Isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice)
Luria-Bertani broth (LB) medium (Merck KGaA, catalog number: 1.10285.0500 )
Equipment
1.5 ml microtube
Ultra-fine-II insulin syringe 1 ml 31 G (0.25 mm x 8 mm) (BD, catalog number: 328820 )
Procedure
Vaccine preparation
Prepare a suspension containing 10 μg/ml of E.coli OMVs in PBS.
Notes:
The total amount of the sample depends on the number of mice in the immunized group. For each mouse, 100 μl of sample will be given. Add extra 100 μl to compensate for any loss of sample during each injection. i.e. If N=5 per group, make a total of 600 μl for each group.
Refer to the protocol " Preparation of Out Membrane Vesicle from Escherichia coli " (Kim et al., 2013b) for preparing OMVs.
Make three aliquots of the above OMVs sample in each 1.5 ml microtube and store at -80 °C until use.
Immunization
At day 0, label the cage (or mouse) to distinguish between immunized and sham.
Bring one of the OMVs sample aliquot to room temperature and mix by vortexing before use.
Hold the mouse in your hand by the dorsal skin so that the head of the mice is pointing the top and its rear legs are down. Maintain the tail with the fingers as in Figure 1 below.
Figure 1. Intraperitoneal injection
Bacteria challenge for induction of sepsis
At day 21, prepare E. coli (5 x 109 CFU/ml) in PBS after culturing E. coli in LB broth for 200 rpm at 37 °C until OD600=1.0.
Note: The total amount of the sample depends on the number of mice that will be challenged. For each mouse, 200 μl of the sample will be given. Add extra 100 μl to compensate for any loss of sample during each injection. i.e. For a total of 10 mice, make 2,100 μl of the sample.
Hold the mouse in your hand by the dorsal skin so that the head of the mice is pointing the top and its rear legs are down. Maintain the tail with the fingers.
Use sterile 1 ml syringe to intraperitoneally inject 200 μl of the E. coli suspension per mouse.
Monitor the survival of mice every 12 h and record.
Note: The symptoms for sepsis will appear after about 6-8 h of challenge. Sacrifice the mice after 5 days of monitoring. Please refer to the reference article for survival results.
Acknowledgments
This protocol was adapted from previously published work (Kim et al., 2013a). The work was supported by a grant from the Korean Ministry of Education, Science and Technology, FPR08B1-240 of the 21C Frontier Functional Proteomics Program and Mid-career Researcher Program of National Research Foundation of Korea (NRF) grant funded by the Korea government MEST (No. 20110000215 and No. 20120005634).
References
Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013a). Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses. J Immunol 190(8): 4092-4102.
Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013b). Preparation of outer membrane vesicle from Escherichia coli. Bio-protocol 3(23): e955.
Article Information
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© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Immunology > Animal model > Mouse
Microbiology > Microbe-host interactions > Bacterium
Microbiology > Microbe-host interactions > In vivo model
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995 | https://bio-protocol.org/en/bpdetail?id=995&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Preparation of Outer Membrane Vesicle from Escherichia coli
Oh Youn Kim
BH Bok Sil Hong
KP Kyong-Su Park
YY Yae Jin Yoon
SC Seng Jin Choi
WL Won Hee Lee
TR Tae-Young Roh
YK Yoon-Keun Kim
Yong Song Gho
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.995 Views: 21727
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Cited by
Original Research Article:
The authors used this protocol in The Journal of Immunology Apr 2013
Abstract
Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. Previous biochemical and proteomic studies have revealed that the Gram-negative bacteria-derived OMVs are composed of various components like outer membrane proteins, lipopolysaccharides, outer membrane lipids, periplasmic proteins, DNA, and RNA. Here, in this protocol, we describe the method to isolate the OMVs from the culture supernatant of Escherichia coli (E. coli).
Keywords: Outer membrane vesicles Bacterial extracellular vesicles Vesicle isolation
Materials and Reagents
Phosphate buffered saline (PBS) (Gibco®, catalog number: 70013-032 )
E. coli (Isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice)
Luria-Bertani broth (LB) medium (Merck KGaA, catalog number: 1.10285.0500 ) (see Recipes)
Equipment
2 L Flasks
Shaking incubator
Centrifuge
500 ml Bottle top filter 43 mm neck (0.45 μm and 0.22 μm) (Corning, catalog number: 430514 , 430513 )
QuixStand Benchtop System (Amersham Biosciences, catalog number: 56-4107-44 )
100-kDa hollow-fiber membrane (Amersham Biosciences, catalog number: 56-4101-33 )
Vacuum pump
Note: All centrifuge tubes and flasks should be autoclaved before use to avoid contamination
Procedure
A single colony of E. coli is transferred to 5 ml of LB broth.
The bacteria are incubated in an orbital bacteria shaking incubator at 200 rpm at 37 °C overnight (8 h).
LB broth of 500 ml is inoculated with 1/100 volume of the overnight cultured cells.
Notes:
Use 2 L flask when culturing 500 ml. Also, since 1/100 volume of 5 ml is 50 μl, before inoculation, increase the volume by adding about 900 μl of fresh LB medium to reduce cell loss.
The yield of OMVs in terms of protein amount is 100 μg per 1 liter of E. coli culture.
The cells are grown for 12 h at 200 rpm at 37 °C.
The cells are pelleted at 5,000 x g for 15 min.
The supernatant fraction is collected and pelleted again at 5,000 x g for 15 min.
The supernatant is collected and filtered through a bottle top filter of pore size 0.45 μm using a vacuum pump.
The filtered supernatant is concentrated to 50-fold by ultra-filtration with a Quixstand Benchtop System using a 100 kDa hollow-fiber membrane.
Note: Because the yield of OMVs is very low, in order to obtain a visible pellet after ultracentrifugation, the total volume of bacteria culture should be more than 5-7 liters. However, depending on the amount of OMV needed, the volume of bacteria culture could be reduced as well as the degree of concentration. Ex. 7 L of bacteria culture supernatant is concentrated to give about 280 ml of the concentrated supernatant to be filled in the total of four ultra-centrifuge tube (70 ml each).
The concentrated supernatant is filtered once again through a 0.22 μm vacuum filter to remove any remaining debris or bacteria.
The resulting filtrate is subjected to ultra-centrifugation at 150,000 x g for 3 h at 4 °C.
The supernatant is removed and the pellet (purified OMV) is resuspended in PBS and stored at – 80 °C until use.
Figure 1. TEM image of E. coli OMV
Recipes
LB medium
1% Tryptone, 0.5% yeast extract, 200 mM NaCl
Acknowledgments
This protocol was adapted from previously published work (Kim et al., 2013). This work was supported by a grant from the Korean Ministry of Education, Science and Technology, FPR08B1-240 of the 21C Frontier Functional Proteomics Program and Mid-career Researcher Program of National Research Foundation of Korea (NRF) grant funded by the Korea government MEST (No. 20110000215 and No. 20120005634).
References
Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013). Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses. J Immunol 190(8): 4092-4102.
Lee, E. Y., Choi, D. S., Kim, K. P. and Gho, Y. S. (2008). Proteomics in gram-negative bacterial outer membrane vesicles. Mass Spectrom Rev 27(6): 535-555.
Park, K. S., Choi, K. H., Kim, Y. S., Hong, B. S., Kim, O. Y., Kim, J. H., Yoon, C. M., Koh, G. Y., Kim, Y. K. and Gho, Y. S. (2010). Outer membrane vesicles derived from Escherichia coli induce systemic inflammatory response syndrome. PLoS One 5(6): e11334.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Kim, O. Y., Hong, B. S., Park, K., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T., Kim, Y. and Gho, Y. S. (2013). Preparation of Outer Membrane Vesicle from Escherichia coli. Bio-protocol 3(23): e995. DOI: 10.21769/BioProtoc.995.
Download Citation in RIS Format
Category
Microbiology > Microbial cell biology > Organelle isolation
Cell Biology > Organelle isolation > Outer membrane vesicles
Biochemistry > Lipid > Extracellular lipids
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Acquisition of Leftward Flow in Xenopus laevis
Thomas Thumberger
MB Martin Blum
Published: Vol 3, Iss 23, Dec 5, 2013
DOI: 10.21769/BioProtoc.996 Views: 9060
Reviewed by: Ru Zhang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Current Biology Jan 2007
Abstract
In Xenopus, the left-right axis is established following an extracellular vectorial leftward flow driven by monocilia at the gastrocoel roof plate (GRP) during late gastrulation / neurulation (Schweickert et al., 2007). As the GRP lies inside the developing archenteron, imaging of flow is challenging. Here we present the detailed procedure to visualize leftward flow in Xenopus laevis embryos.
Keywords: Cilia Left-right asymmetry Imaging Leftward flow Gastrocoel roof plate
Materials and Reagents
Fluorescent micro beads (e.g. FluoSpheres 0.5 μm) (Life Technologies, catalog number: F-8813 )
Agarose
Penicillin/Streptomycin (10.000 Units/ml; Gibco®, catalog number: 15140 )
HEPES Pufferan (Carl Roth, catalog number: HN78.3 )
Bead solution (see Recipes)
5x MBSH (Modified Barth‘s Saline) (see Recipes)
Equipment
Sharp forceps (Fine Science Tools, catalog number: Dumont #5 )
Micro blade (Fine Science Tools, Dissecting Knife - Fine Tip)
Sectioning dish (Petri Dish with bottom covered in 1% Agarose in 1x MBSH)
Transfer pipet (Carl Roth, model: EA65.1 )
Glass staining block (Karl-Hecht Assistent (Lymphbecken) 2020)
Microscope slides
Coverslips
5 ml syringe
Vaseline
Fluorescence microscope equipped with a digital camera (wide field)
Large Petri dish filled with PBS or MBSH for explant retrieval
Procedure
Raise Xenopus embryos to stage 17 (Nieuwkoop et al., 1994).
Build a flexible imaging chamber (Figure 1) by putting the opening of a vaseline filled syringe directly onto a microscope slide. Gently press the piston to release vaseline as you draw a rectangle (approximately 1.5 x 1 cm) onto the slide. Make sure that the rectangle is closed. Fill the rectangle with the bead solution until it has a convex meniscus.
Figure 1. A flexible imaging chamber
Cut dorsal explant (cf. Figure 2 in Blum et al., 2009):
Transfer a stage 17 embryo into a sectioning Petri dish filled with 1x MBSH.
With a micro blade, remove the head by transversally cutting the anterior part of the embryo.
Erect the cup-shaped embryo such that you can see inside. Then place small alternating cuts at the left and right ‘lateral lines’ until you reach the area of the circumblastoporal collar.
With forceps and the micro blade, pull apart the dorsal and ventral halves of the embryo until you see the opening of the blastopore from the inside.
Place a final cut ventral to the blastopore to separate the dorsal explant from the rest of the embryo. Make sure not to touch the dorsal explant with the forceps or blade from the inside.
You should now be left with a bathtub shaped dorsal explant with the GRP placed at its deepest point.
With a transfer pipet, carefully bring the dorsal explant into the glass staining block that contains the FluoSphere bead solution and gently pipet the explant up and down in this medium to make sure that FluoSpheres reach the GRP.
Transfer the dorsal explant into the FluoSphere solution filled chamber by carefully pipetting. Make sure that the explant never touches the surface of any liquid as surface tension disintegrates the tissue. Orient the explant with the inside facing up, i.e. towards you.
Seal the flexible chamber with a coverslip by gently pushing it down with opened forceps. Carefully push until the coverslip touches the rim of the dorsal explant, i.e. the left and right ‘lateral line’ and the ventral side of the circumblastoporal collar.
While pushing down, sometimes the ventral circumblastoporal collar folds over and blocks the GRP from view. If that happens, a slight drag of the coverslip towards the posterior pole can shear the explant to allow imaging of the GRP.
Let the setup rest for 5-10 min to allow the explant to deform, which otherwise would suggest false particle movements during early acquisition.
Put the slide in your (wide-field) fluorescence microscope and focus on the GRP.
Excite with the required wavelength of the FluoSpheres - you should see a ‘starry sky’. Now adjust focus to the focal plane slightly above the GRP.
Note: as the GRP cilia are just 5 μm in length, leftward flow occurs only in the plane right above the epithelium (Schweickert et al., 2007). To facilitate finding the right plane, focus into the tissue until you do not see fluorescence of any bead. Now carefully change the focal plane until the first beads appear in focus.
In wild type embryos, you should see particles moving to the left of the GRP. In Xenopus laevis, leftward flow is relatively slow (~2.5 μm/s) and therefore best visualized using time-lapse videography (Schweickert et al., 2007). Depending on further analysis and hence temporal resolution, acquire a movie with at least 2 fps.
For retrieving the dorsal explant after investigation, submerge the complete slide in a large Petri dish filled with 1x MBSH or 1x PBS and carefully remove the coverslip with forceps. The explant should float into the buffer from which it can be pipetted into fixative for further analysis (in situ hybridization, immunohistochemical staining, etc.).
Recipes
Bead solution
Dilute FluoSpheres 1:2,500 in 1x MBSH (prepare ~5 ml)
5 x MBSH (1 L)
Note: Before usage, dilute to 1x with H2O
25.7 g NaCl
0.375 g KCl
1 g NaHCO3
1 g MgSO4.7H2O
0.39 g (CaNO3)2.4H2O
0.3 g CaCl2.2H2O
11.9 g Hepes
5 ml penicillin/streptomycin
Acknowledgments
This work was supported by Deutsche Forschungsgemeinschaft grants to M.B.
References
Blum, M., Beyer, T., Weber, T., Vick, P., Andre, P., Bitzer, E. and Schweickert, A. (2009). Xenopus, an ideal model system to study vertebrate left-right asymmetry. Dev Dyn 238(6): 1215-1225.
Nieuwkoop, P. and Faber, J. Normal Table of Xenopus laevis (Daudin), 1994, GARLAND PUBLISHING, New York&London, USA.
Schweickert, A., Weber, T., Beyer, T., Vick, P., Bogusch, S., Feistel, K. and Blum, M. (2007). Cilia-driven leftward flow determines laterality in Xenopus. Curr Biol 17(1): 60-66.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Thumberger, T. and Blum, M. (2013). Acquisition of Leftward Flow in Xenopus laevis. Bio-protocol 3(23): e996. DOI: 10.21769/BioProtoc.996.
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Category
Developmental Biology > Morphogenesis > Motility
Cell Biology > Cell imaging > Fluorescence
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997 | https://bio-protocol.org/en/bpdetail?id=997&type=0 | # Bio-Protocol Content
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Invadopodia Detection and Gelatin Degradation Assay
BD Begoña Díaz
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.997 Views: 22312
Reviewed by: Lin FangFanglian He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Apr 2013
Abstract
This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia and their activity is simultaneously assessed and quantified by a fluorescent microscopy-based assay.
Keywords: Cancer Invasion Invadopodia Metastasis Gelatin
Materials and Reagents
Cells (i.e. SCC61 head and neck carcinoma cells)
Tissue culture media (i.e. DMEM) (Mediatech, catalog number: 10013CV )
Sterile PBS
Trypsin 0.05%/EDTA (Life Technologies, catalog number: 25300054 )
Penicillin/Streptomycin solution (Omega Scientific, catalog number PS-20 )
Ethanol (Decon Labs, catalog number: 2801 )
Forceps Dumont #5 (Fine Science Tools, catalog number: 11252-20 )
25% Glutaraldehyde (Polysciences, catalog number: 00376-500 )
Sucrose (Fisher Scientific, catalog number: S3500 )
Sodium Borohydride (Fisher Scientific, catalog number: S67825 )
16% Formaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
Gelatin from pig skin, Oregon green 488 conjugate (Life Technologies, catalog number: G13186 )
Alexa Fluor 568 Phalloidin (Life Technologies, catalog number: A12380 )
Mounting medium, Vectashield with DAPI (Vector Laboratories, catalog number: H-1200 )
Bovine Serum Albumin fraction V (EMD Millipore, catalog number: 2980-1KG )
Triton X-100 (Promega corporation, catalog number: H5141 )
Parafilm (Bemis Company, catalog number: PM996 )
Equipment
Round 18 mm diameter glass coverslips (0.13-0.17 mm thickness) (Carolina Biological Supply Company, catalog number: 633033 )
Microscope glass slides (75 x 25 mm) (Thermo Fisher Scientific, catalog number: 1255015 )
12-well Microplates (Corning, catalog number: 3513 )
Thermomixer or water bath set at 60 °C
Tissue culture incubator
Hemocytometer (LabSource, catalog number: 0267154 ) http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/counting-cells-in-a-hemacytometer.html
Fluorescence Microscope
Vacuum source
Procedure
Preparing fluorescent gelatin-coated coverslips
The objective of this step is to perform a gelatin coating as homogeneous as possible. To achieve that, it is better to coat no more than 4 coverslips at a time, repeating the process as many times as necessary to obtain the desired final number of coverslips. By doing that, the fluorescent gelatin stock solution is used recently warmed for each group of four coverslips. Also, it is important to work reasonably quickly (through steps A1 to A4 in Figure 1) to avoid that the gelatin starts drying before it has formed a thin uniform coating of the coverslip.
Sterilize coverslips by soaking them in 70% Ethanol solution, let them air dry. Gelatin-coated coverslips can be prepared in batches of 12 or 24 (one or two multiwell plates) and stored in the dark at 4 °C for up to 1-2 weeks.
Prepare fluorescent gelatin solution by diluting the gelatin stock (prepared according to manufacturer’s instructions at 1 mg/ml by adding 5 ml of distilled water to the commercial vial), in PBS containing 2% sucrose. Typically, gelatin is used at a final concentration of 0.2 mg/ml. Warm the fluorescent gelatin stock at 60 °C prior to dilution and keep the working solution protected from light and warmed during the coating process. Prepare around 500 μl of fluorescent gelatin working solution for 24-coverslip batch. Diluted fluorescent gelatin solution can be reused once upon frozen storage.
Cut a piece of parafilm of around 4 x 2 inches and tape it to the bench top. This will facilitate quick manipulation of the coverslips. Place a set of 4 coverslips in line on top of the parafilm. Have the gelatin working solution pre-warmed in-hand (i.e. by having a thermomixer or a water bath by your side on the bench top). Pipet 100 μl of warmed fluorescent gelatin solution on top of the first coverslip and return the stock to 60 °C (step A1 in Figure 1). Using forceps, lift the coverslip with the gelatin on top and, keeping it horizontal, use the other hand to spread the gelatin with the outside of the same pipet tip attached to a pipettor (step A2 in Figure 1). When spread, hold this coverslip over the next one to prepare and tilt the coverslip to a vertical position letting the excess of gelatin to fall on the next coverslip (step A3 in Figure 1). Still holding the first coverslip in a vertical position, use a soft vacuum source (only turned on lightly, just enough to hold a pipet tip at the end of the hose) to aspirate the extra-coating left on the bottom edge of the coverslip (step 4 in Figure 1). Using stronger vacuum (completely turned on) is not advised since it will leave a gelatin layer that is too thin. Place the coverslip inside the well of a 12-well plate protected from light. Immediately proceed to repeat the same procedure with the next coverslip.
Figure 1. Steps for coating coverslips with fluorescent gelatin. 1-Add pre-warmed gelatin solution to the tip of sterilized coverslips; 2-Hold coverslip with forceps and spread gelatin with the help of a pipet tip. 3-Remove the excess gelatin solution over the next coverslip to prepare. 4-Carefully aspirate the excess gelatin from the lower side of the coverslip. Refer to text for further details.
When all coverslips are coated, let them dry (coating will look whitish when dry).
Add 1 ml of a pre-chilled solution of glutaraldehyde (diluted from the stock solution at 0.5% in PBS) to each well and incubate for 15 min on ice.
Remove the glutaraldehyde and dispose properly. Wash coverslips three times at room temperature with PBS.
Add 1 ml of a freshly prepared solution of Sodium Borohydride (5 mg/ml in PBS) and incubate for 3 minutes at room temperature. Stir the plate if necessary to avoid that the Hydrogen bubbles lift the coverslips to the surface of the liquid.
Remove the Sodium Borohydride and dispose properly. Wash coverslips three times at room temperature with PBS.
Transfer plates to a tissue culture hood. Using the forceps (an old pair of forceps with a bent tip may help in this process), transfer coverslips to a sterile 12-well plate and wash three times with sterile PBS. Coverslips may be used the same day or stored in PBS containing 200 units per ml of penicillin and 200 μg per ml of streptomycin (1:50 from the stock solution) at 4 °C in the dark for up to 2 weeks.
Notes:
An alternative method to cover the glass coverslips with the gelatin solution consists on pipetting the gelatin over the parafilm and invert the glass coverslip over the gelatin. This method may not achieve the same homogeneity but it may be easier for manipulation of coverslips.
Using up to 1 mg/ml of fluorescently labeled gelatin solution to coat glass coverslips might help to increase the number of invadopodia formed by certain cell lines. Some cells attach very strongly and “pull” the gelatin coating in addition to degrading it. The effect of pulling cannot be distinguished from the effect of degradation for quantification purposes on this assay. Using 1 mg/ml of fluorescently labeled gelatin solution to coat glass coverslips or decreasing FBS in the growing medium might decrease the ability of some cells to “pull” the gelatin coating.
Performing the fluorescent gelatin degradation assay
Working under sterile conditions, transfer the desired number of coated coverslips to a new sterile 12-well plate. If the fluorescent gelatin coated coverslips were already stored in antibiotic-containing PBS, wash them three times with sterile PBS and incubate in 1 ml of the same complete growth medium that you will use in the experiment. Place them inside the 37 °C tissue culture incubator.
Collect the cells to use in the assay using standard techniques (i.e. trypsinization), and count them using the hemocytometer or other suitable method.
Plate 1 ml of a cell suspension, typically containing between 20-40,000 cells on top of each well containing 1 ml of medium.
Return cells to the incubator for 8-16 h. The length of the assay should be determined empirically for each cell line and conditions.
Processing cells for invadopodia detection and fluorescent microscopy
At endpoint, wash cells once with PBS and quickly fix in a formaldehyde solution (4% in PBS) for 10-15 minutes at room temperature and protected from light.
Remove formaldehyde and dispose properly. Wash three times with PBS and incubate in a BSA solution (3% in PBS containing 0.1% Triton X-100) for 15-30 min at room temperature and protected from light.
Remove BSA solution and stain F-actin with Alexa Fluor 568 Phalloidin (diluted around 1:500 to 1:100 in PBS containing 0.3% BSA and 0.1% Triton X-100). Incubate for 0.5 to 1 h at room temperature protected from light. The optimal phalloidin dilution and incubation time depends on the cell line used and should be determined empirically.
Remove the phalloidin solution and wash three times with PBS containing 0.1% Triton-X100 and three times with PBS alone.
Mount the coverslips by inverting them over a glass slide containing a drop of mounting medium containing DAPI. Slides can be stored in the dark at 4 °C for several weeks.
Imaging and quantifying invadopodia formation and activity
Slides can be imaged with a fluorescent microscope equipped for detection of Alexa 488, Alexa 568 and DAPI. Invadopodia are detected in the “red” channel as F-actin rich puncta in the ventral surface of the cell in contact with the gelatin (Figure 2 C). Gelatin degradation is detected in the “green” channel as dark areas over the green background (Figure 2 B). Typically, a subset of F-actin rich structures will co-localize with discrete gelatin degradation spots, indicating that these F-actin rich structures are likely invadopodia (Figure 2 A).
Quantification of invadopodia formation and activity may be performed on the same set of digital images. To perform a representative image collection, it is advisable to image areas that are representative to the whole coverslip in order to account for differences in the thickness of the gelatin coating. A possibility is to divide the coverlip into three imaginary columns and image five rows per column covering the whole height of each column. At least 15 fields per coverslip should be imaged, typically under 40x magnification taking all three channels (“red”, “green” and “blue”). Moving from one field to the next when in the channel for DAPI will help you identify areas containing similar number of cells while you are “blinded” for any other experimental outcome in a particular field to be imaged.
Figure 2. Representative images of invadopodia and gelatin degradation assay. Digital images from a gelatin degradation assay performed on SCC61 cells. Images were obtained using a 40x objective in a fluorescence microscope. A. Merged channels showing fluorescent gelatin (green), F-actin staining (red) and nuclei (blue). B. Green channel (gelatin). C. Red channel (F-actin). D. Blue channel (nuclei). Arrowheads point to invadopodia in A and C.
To quantify invadopodia formation, count the number of cells forming invadopodia on each digital image and normalize to the number of total cells in that same image. After counting all your images, collected data may be represented as “percent of cells forming invadopodia”. Perform the appropriate statistical analysis to determine significant differences among treatments.
To quantify invadopodia activity, black and white images of gelatin degradation are analyzed using ImageJ (NIH). The objective is to measure “area fraction” (the percent of area that corresponds to degradation) on a given image. The “area fraction” value will then be normalized to the number of nuclei in each image as measured from the DAPI channel in the same field (Figure 3). The steps for obtaining the “area fraction” value are:
Set Image J to measure “area fraction”: Analyze>Set Measurements>select “Area Fraction”.
Open a black and white image of gelatin degradation (green channel) in Image J and then go to Image>Adjust>Threshold. If the black areas over white background are representative of the degradation in the original image, no further adjustment is necessary. If the program detects black areas that are broader than the actual degradation (i.e. because of irregular gelatin coating), the threshold has to be manually adjusted. On the new window move the bottom on the lower bar towards the left until the dark areas are representative of the real degradation observed in the original image.
When the final image is representative of the gelatin degradation on the original image (as in Figure 3 C) click “set”. Measure “area fraction” by: Analyze>Measure. The values obtained in Image J may be exported to Microsoft Excel.
The measurements corresponding “to area fraction” are normalized to the number of nuclei in the DAPI channel image from the same field. The final value corresponds to “normalized degradation”. The same analysis is repeated for each image of each sample. Values can be represented as mean and standard deviations. Perform the appropriate statistical analysis to determine significant differences among treatments. An example is showed in Figure 3.
Note: When assessing the formation of invadopodia in your cell line of interest for the first time, it is advisable to perform additional immunofluorescent experiments to assess the presence of invadopodia components such as Tks5, cortactin and Arp2/3 at the F-actin rich structures.
Figure 3. Quantification of invadopodia activity. Images from the green channel (gelatin) and blue channel (nuclei) from the same microscopy field are used. The original image from the green channel (A) is processed using Image J to obtain the image in (B), and the area fraction value. The original image from the blue channel (C) is used to calculate cell number. These two values are used to obtain the“normalized degradation” value of each microscopy field from a sample.
Acknowledgments
This protocol was initially adapted from: Mueller et al. (1992). The first adaptation was published in: Berdeaux et al. (2004). It was later implemented in: Díaz et al. (2013). Funding during first adaptation: National Institutes of Health grant CA17542 to G.S. Martin. Funding during implementation: National Institutes of Health CA129686 to S. A. Courtneidge. Other Acknowledgments: The author would like to acknowledge Dr. G.S. Martin (UC Berkeley, California) and S.A. Courtneidge (UHSU, Portland, Oregon) for their support over the time this protocol was adapted and implemented.
References
Berdeaux, R. L., Diaz, B., Kim, L. and Martin, G. S. (2004). Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function. J Cell Biol 166(3): 317-323.
Diaz, B., Yuen, A., Iizuka, S., Higashiyama, S. and Courtneidge, S. A. (2013). Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia. J Cell Biol 201(2): 279-292.
Mueller, S. C., Yeh, Y. and Chen, W. T. (1992). Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells. J Cell Biol 119(5): 1309-1325.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Díaz, B. (2013). Invadopodia Detection and Gelatin Degradation Assay. Bio-protocol 3(24): e997. DOI: 10.21769/BioProtoc.997.
Diaz, B., Yuen, A., Iizuka, S., Higashiyama, S. and Courtneidge, S. A. (2013). Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia. J Cell Biol 201(2): 279-292.
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Category
Cancer Biology > General technique > Cell biology assays > Cell migration
Cancer Biology > Invasion & metastasis > Cell biology assays > Cell invasion
Cell Biology > Cell imaging > Fluorescence
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998 | https://bio-protocol.org/en/bpdetail?id=998&type=0 | # Bio-Protocol Content
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Ex utero Electroporation into Mouse Embryonic Neocortex
BN Branden R. Nelson
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.998 Views: 11388
Reviewed by: Xuecai Ge Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience May 2013
Abstract
This technique allows highly efficient and reproducible transfer of DNA/RNA into the embryonic neocortex of rodents across multiple ages. Ex utero electroporation compliments the more technically difficult in utero electroporation technique by maximizing the number of embryos for available for a given experiment, as well as increasing the variety of constructs used in each experiment, thereby helping to reduce their overall numbers. Ex utero electroporation followed by short term organotypic slice culture of embryonic brain sections allows immediate access to multiple slices for choosing optimal ones for live-cell imaging experiments, and characterization of various NSC manipulations in the intact stem cell niche. (see also “In utero Electroporation of Mouse Embryo Brains” (Ge, 2012); “Organotypic Slice Culture of Embryonic Brain Sections” (Calderon de Anda, 2013). Additionally, ex utero electroporated neocortices can be used for in vitro primary cell cultures with further dissection, dissociation into single cells, and plating on cover slips or in multi-well dishes according to standard techniques: note this procedure can be performed immediately after electroporation, prior to the onset of ectopic gene expression, or after overnight slice culturing to collect just the region of electroporated cells.
Materials and Reagents
Timed embryonic mice embryos: A wide range of developmental timepoints work very well for this procedure, from E10-18.5. Note however that earlier embryos (E10-12.5) are very delicate and may require modified vibratome slicing techniques and/or manual cutting with a micro-knife, which can be embedded in collagen 3D-gels (to help preserve tissue structure) and cultured on Millicell inserts, or floated as whole brain explants overnight in a multi-well plate on a nutator/rotator. Likewise, older embryos E17-18 will have more developed skin and craniums, requiring a bit more force to inject DNA solution into the ventricles, which are becoming smaller relative to the size of the animal.
DNA prepared at (≥1 μg/μl): Qiagen Maxi Prep kit (QIAGEN, catalog number: 12663 ) or EndoFree Plasmid Kit (QIAGEN, catalog number: 12362 )
Fast green FCF (Sigma-Aldrich, catalog number: F7252 )
HBSS (Life Technologies, catalog number: 14025-126 )
4% Low-melting point agarose (in sterile 1x PBS or 1x HBSS)
Low-Melting point agarose (GeneMate Sieve GQA Agarose, catalog number: E-3112 )
Ice
Kimwipe
Equipment
35 mm dishes
Multi-well cell culture dish (12 to 24 wells)
Electroporator (BTX The Electroporation Experts, model: ECM 830 Square Wave Electroporation System)
3 mm-paddle Platinum Tweezertrodes (BTX The Electroporation Experts, model: 45-0487 )
Footswitch for ECM 830 (BTX The Electroporation Experts, model: 1250FS )
Mouth pipette (Sigma-Aldrich, catalog number: P0799-1PAK )
Micropipettes (Borosilicate with filament O.D.: 1 mm, I.D.: 0.78 mm, 10 cm length) (SUTTER INSTRUMENT, catalog number: BF100-78-10 )
Micropipette puller P-97/IVF (SUTTER INSTRUMENT)
Dissecting stereoscope with flexible lighting
Dumont forceps (#5 and #55) (Fine Science Tools)
Disposable plastic transfer pipettes
Sterile scalpel
Fluorescent dissecting stereoscope for checking transfections
Digital camera (if necessary) (OLYMPUS, model: MVX10 )
Procedure
Collect timed embryonic mice embryos according to approved protocols at your institution. Hold embryos in ice-cold HBSS filled dish until ready for use.
Pour melted agarose into a 35 mm dish ~1/2 to 2/3 full, and let solidify over ice.
Cut a small trapezoid out of the center of the dish with a scapel, discard (Figure 1A).
Fill this dish with HBSS to almost full (Figure 1A).
Transfer single embryo into the well of this dish using cut transfer pipette or forceps (Figure 1A).
Fill micropipette with DNA solution mixed with enough Fast Green to just visualize.
Position embryo in well, and hold in place with forceps while micro-injecting ~0.2-0.5 μl DNA into the lateral ventricle. Flip embryo over, reposition, and microinject DNA into the contralateral ventricle.
Note: Each ventricle should be visualized due to colored solution (Figure 1A).
Hold tweezertrodes over the head in manner to target current into the dorsal neocortex.
Note: The targeted position is highly dependent on the orientation of the tweezertrodes. For example, holding the positive electrode over the dorsal neocortex on one side of the brain means that the negative electrode will be placed in a more ventral/diagonal position, slightly under the contralateral jaw. This is most easily accomplished by using another pair of forceps in the other hand (i.e. left), while holding tweezertrodes in the other hand (i.e. right).
Electroporate embryo using the remote footswitch: 3–5 pulses, 35 mV, 100 ms intervals.
Flip embryo over, reposition tweezertrodes accordingly, and re-electroporate the contralateral neocortex using the same procedure.
Notes:
Varying electrode placement can target other specific regions, including the hippocampus/Dentate, ventral telencephalon, as well as other regions that contain a lumen.
During electroporation, it is best to not contact the embryo directly.
Transfer electroporated embryo into multi-well dish placed on ice with HBSS to recover using cut transfer pipette/forceps.
Clean electrodes after each embryo electroporation by wiping with dry Kimwipe.
Repeat procedure until all embryos have been electroporated.
Note: Multiple different DNA constructs and combinations can be used within this ex utero procedure.
Dissect the brain from each embryo, and transfer into ice-cold HBSS in new multi-well plate until all brains have been removed.
Note: In some cases the meninges could be removed to help vibratome sectioning depending on the actual age of embryos, younger embryos are more delicate (E10-12).
Embed brains in 4% low-melting agarose and prepare for vibratome sectioning and organotypic brain slice culture as described in (see Organotypic Slice Culture of Embryonic Brain Sections (Calderon de Anda, 2013).
Slices can be visualized the following day with a fluorescent stereoscope. With practice, multiple slices should contain fluorescent cells in each neocortex. For example, Figure 1B shows typical example of RFP expressing plasmid targeted in to each neocortex in a wild-type embryo. Figure 1C,D is example of RFP targeted into each neocortex of a transgenic GFP expressing animal (Nelson et al., 2013).
Figure 1. Ex utero electroporation in embryonic mouse neocortex. A. Schematic of technique; B. Example of double electroporated RFP plasmid targeted to each neocortex in wild type E14.5 mice cultured overnight; C. Example of double electroporated RFP plasmid targeted to each neocortex in transgenic Tbr2GFP E14.5 neocortex; D. High power view. Black and red represent negative and positive electrodes, respectively, so keep in mind their orientation since it controls direction and targeting: green represents the Fast Green colored DNA solution that fills the lateral ventricles, revealing their shape, and indicates a successful fill.
Note: Images were acquired with a fluorescent stereoscope equipped with GFP/RFP filter sets and digital camera (Olympus, MVX10)
Acknowledgments
This protocol was adapted from Nelson et al. (2013), and was supported by NIH Grants R21 MH087070 and RO1 MH080766-S to R.F.H.
References
Calderon de Anda, F. (2013). Organotypic slice culture of embryonic brain sections. Bio-protocol, 3(3): e327.
Ge, X. (2012). In utero electroporation of mouse embryo brains. Bio-protocol 2(13): e231.
Nelson, B. R., Hodge, R. D., Bedogni, F. and Hevner, R. F. (2013). Dynamic interactions between intermediate neurogenic progenitors and radial glia in embryonic mouse neocortex: potential role in Dll1-Notch signaling. J Neurosci 33(21): 9122-9139.
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Neuroscience > Development > Morphogenesis
Molecular Biology > DNA > Transformation
Cell Biology > Tissue analysis > Tissue isolation
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999 | https://bio-protocol.org/en/bpdetail?id=999&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
ImmunoFISH for Adherent Cultured Mammalian Cells
FR Francesca Rossiello
MF Marzia Fumagalli
FF Fabrizio d’Adda di Fagagna
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.999 Views: 11388
Reviewed by: Lin Fang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Cell Biology Apr 2012
Abstract
This protocol is optimized for immunoFISH staining of adherent cultured mammalian cells. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) and FISH against telomeric DNA.
Keywords: ImmunoFISH DNA damage response Telomeres
Materials and Reagents
Cells
4% PFA
Methanol/acetone 1:1
TritonX100
Primary antibody : 53BP1 #NB 100-304 rabbit from Novus
Second antibody: goat anti-rabbit Alexa Fluor® 488 Dye
PBS
Glycine
Fish gelatin (Sigma-Aldrich, catalog number : G7041 )
BSA
Formamide
Tris HCl, pH 7.4
Telomeric PNA probe (TelC-Cy3 from PANAGENE, catalog number: F1002-5 )
DAPI
Mowiol 4-88 reagent (Calbiochem®)
PBG (see Recipes)
Hybridization mixture (see Recipes)
Blocking reagent (Roche Diagnostics, catalog number: 11096176001 ) (see Recipes)
Wash solution I (see Recipes)
Wash solution II (see Recipes)
Equipment
Glass coverslips
12 multiwell plate
Metal thermoblock
Humidified chamber
Procedure
Grow cells on glass coverslips (e.g. BJ normal human fibroblasts).
Transfer the coverslip to a 12 multiwell plate.
Wash briefly with 1x PBS.
Fix with either 4% PFA, 10 min, RT or methanol/acetone 1:1, 2 min, RT (it depends on the antibody, does not affect the FISH signal; use methanol/acetone for 53BP1 staining).
Wash with 1x PBS, 3 times, 5 min.
Only for PFA-fixed cells, incubate with 0.2% TritonX100 in PBS, 10 min, then wash with 1x PBS, 3 times, 5 min.
Block with 1x PBG, 1 h, RT.
Incubate with primary antibody diluted in 1x PBG, 50 μl for each coverslip. Incubation time depends on the antibody, most work in 1 h, RT, or overnight at 4 °C. (For 53BP1 dilute 1:200 and incubate 1 h at RT).
Wash with 1x PBG, 3 times, 5 min.
Incubate with secondary antibody diluted in 1x PBG, 45 min, RT.
Wash with 1x PBG, twice, 5 min.
Wash with 1x PBS, twice, 5 min.
Re-fix cells with PFA 4% + triton 0.1%, 10 min RT (use PFA also if you have previously fixed cells with methanol/acetone).
Incubate with glycine 10 mM in H2O, 30 min, RT.
Wash with 1x PBS, 3 times, 5 min.
Prepare the hybridization mixture and put 20 μl on a glass slide for each coverslip.
Transfer the coverslip carefully on the drop without making bubbles.
Put the slide directly on a metal thermoblock at 80 °C, 5 min.
Hybridize in a humidified chamber, 2 h, RT.
Remove coverslip from the slide and put it back in the 12 wells plate.
Wash with Wash solution I, twice, 15 min.
Wash with Wash solution II, 3 times, 5 min.
Incubate with DAPI, 2 min, RT.
Wash briefly with 1x PBS.
Mount with mowiol.
Store the slides at 4 °C for short time storage (2 weeks) or at -20 °C. It is recommended to analyze the fluorescence as soon as possible to avoid fluorophore fading.
Figure 1. An image of ImmunoFish stained human fibroblasts cells. DAPI is in blue, 53BP1is in green and telomeric PNA probe is in red.
Recipes
10x PBG (prepare 5 ml aliquotes and store them in 50 ml tubes at -20 °C, the day of immunoFISH dilute them in 1x PBS)
Fish gelatin
2%
BSA
5%
1x PBS
to volume
Hybridization mixture (always prepare fresh)
Formamide
70%
Blocking reagent
1x
Tris HCl pH 7.4
10 mM
Telomeric PNA probe
0.5 μM
H2O
to volume
10x Blocking reagent
Prepare small aliquots and store them at -20 °C
Wash Solution I (250 ml) (always prepare fresh)
Formamide
175 ml
BSA 10%
2.5 ml
Tris HCl 1 M pH 7.4
2.5 ml
H2O
to volume
Wash Solution II (350 ml) (always prepare fresh)
Tris HCl 1 M pH 7.4
35 ml
NaCl 5 M
10.5 ml
Tween 20 10%
2.5 ml
H2O
to volume
Acknowledgments
The F.d’A.d.F. laboratory is supported by FIRC (Fondazione Italiana per la Ricerca sul Cancro), AIRC (Associazione Italiana per la Ricerca sul Cancro), European Union (GENINCA, contract number 202230), HFSP (Human Frontier Science Program), AICR (Association for International Cancer Research), EMBO Young Investigator Program and Telethon.
References
Fumagalli, M., Rossiello, F., Clerici, M., Barozzi, S., Cittaro, D., Kaplunov, J. M., Bucci, G., Dobreva, M., Matti, V. and Beausejour, C. M. (2012). Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat Cell Biol 14(4): 355-365.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Rossiello, F., Fumagalli, M. and di Fagagna, F. D. (2013). ImmunoFISH for Adherent Cultured Mammalian Cells. Bio-protocol 3(24): e999. DOI: 10.21769/BioProtoc.999.
Download Citation in RIS Format
Category
Cell Biology > Cell structure > Chromosome
Cell Biology > Cell imaging > Fluorescence
Biochemistry > Protein > Immunodetection > Immunostaining
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