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1,095 | https://bio-protocol.org/exchange/protocoldetail?id=1095&type=0 | # Bio-Protocol Content
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Native BAD-1 Binding to Heparin-agarose
TB T. Tristan Brandhorst
Published: Vol 4, Iss 7, Apr 5, 2014
DOI: 10.21769/BioProtoc.1095 Views: 7046
Edited by: Fanglian He
Reviewed by: Kanika Gera
Original Research Article:
The authors used this protocol in Jul 2013
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Abstract
BAD-1 is an adhesin created by the dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis. We have determined that it has an affinity for heparin, which may explain its impact on virulence and human immune function as a number of cells related to immune function have heparin like moieties on their surfaces. This assay allows a quantification of binding between soluble BAD-1 and immobilized heparin.
Materials and Reagents
Heparin-agarose resin (Sigma-Aldrich, catalog number: H6508 ) (prior to use it is washed 3x in five volumes of tricine buffer to eliminate free heparin)
10 μg of BAD-1 [purified according to the method of Brandhorst et al. (2005)]
25 microliters of soluble medical-grade sodium heparin for injection (50 mg/ml) (Elkins-Sinn Inc)
Tricine buffer (Sigma-Aldrich, catalog number: T0377 ) (see Recipes)
Equipment
Accuspin micro17 microcentrifuge (Thermo Fisher Scientific)
Nanodrop ND1000 spectrophotometer
Procedure
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Copyright: © 2014 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:
Brandhorst, T. T. (2014). Native BAD-1 Binding to Heparin-agarose. Bio-protocol 4(7): e1095. DOI: 10.21769/BioProtoc.1095.
Taguchi, Y., Mistica, A. M., Kitamoto, T. and Schätzl, H. M. (2013). Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. PLoS Pathog 9(6): e1003466.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Interaction
Biochemistry > Protein > Activity
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1,096 | https://bio-protocol.org/exchange/protocoldetail?id=1096&type=0 | # Bio-Protocol Content
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Seed Coat Ruthenium Red Staining Assay
HM Heather E. McFarlane
DG Delphine Gendre
TW Tamara L. Western
Published: Vol 4, Iss 7, Apr 5, 2014
DOI: 10.21769/BioProtoc.1096 Views: 19168
Edited by: Ru Zhang
Reviewed by: Renate Weizbauer
Original Research Article:
The authors used this protocol in Jul 2013
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Abstract
The goal of this protocol is to assay for defects in synthesis/secretion/release of seed coat mucilage by ruthenium red staining of mature whole seeds.
The mucilage secretory cells of the Arabidopsis seed coat synthesize and secrete a large quantity of primarily pectinaceous mucilage to a ring-shaped apical domain during their differentiation. This makes them an excellent model system to identify genes involved in both cell wall synthesis and secretion (et al., 2000). When wild-type seeds are incubated in ruthenium red stain, hydrated mucilage is extruded from epidermal cells and a ‘halo’ of red-stained mucilage is observed surrounding the seed (Western et al., 2000). Reduced mucilage staining may result from defects in cell wall biosynthesis, secretion, or impaired release upon hydration.
Keywords: Arabidopsis Seed coat Mucilage Ruthenium red Pectin
Materials and Reagents
Seeds
0.01% ruthenium red solution (Sigma-Aldrich, catalog number: 11103-72-3 )
50 mM EDTA (optional)
dH2O (pH 6.5-7.5) [buffer with 10 mM Tris (pH 7.6), if necessary]
Equipment
Microfuge tubes
Transfer pipet or aspirator
Orbital shaker
White porcelin depression plate or clean depression slides
Dissecting or compound microscope
Procedure
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Category
Plant Science > Plant physiology > Tissue analysis
Biochemistry > Carbohydrate > Glycoprotein
Cell Biology > Tissue analysis > Tissue staining
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1,097 | https://bio-protocol.org/exchange/protocoldetail?id=1097&type=0 | # Bio-Protocol Content
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Isolation and Immortalization of Fibroblasts from Different Tumoral Stages
Fernando Calvo
SH Steven Hooper
ES Erik Sahai
Published: Vol 4, Iss 7, Apr 5, 2014
DOI: 10.21769/BioProtoc.1097 Views: 18824
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Jun 2013
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Abstract
Tumour microenvironment and cancer-associated fibroblasts in particular exhibit tumour promoting abilities that are not present in their normal counterparts (Calvo et al., 2013; Hanahan and Coussens, 2012). Therefore, functional and molecular characterization of the modifications occurring in fibroblasts during tumour progression is essential to fully understand their role in tumour progression. Previous studies have addressed this issue using human fibroblasts and comparing normal and adjacent fibroblasts to tumour-associated fibroblasts (Kalluri and Zeisberg, 2006). However, these studies are hampered by the intrinsic variability of human samples (e.g. pairing, age, genomic landscape, etc). In order to overcome these issues, we used a fully characterised mouse breast cancer model, MMTV-PyMT (Guy et al., 1992; Lin et al., 2003). MMTV-PyMT transgenic mice express the Polyoma Virus middle T antigen under the direction of the mouse mammary tumor virus promoter/enhancer. This is a multifocal luminal breast cancer model that goes through well defined and characterised stages (namely, hyperplasia, adenoma, carcinoma and invasive carcinoma). Interestingly, this model has a 100% incidence, is very desmoplastic (presenting high concentration of fibroblasts) and gives raise to spontaneous metastasis in the lung with 80-94% incidence. Importantly, at least for the inguinal mammary glands (glands 4 and 9), the different tumoral stages are well correlated to the age of the mouse: hyperplasia arising at 6 weeks of age, adenoma between 6-8 weeks of age, carcinoma and invasive carcinoma from 8 weeks onwards. This model allowed us to confidently isolate fibroblasts from different tumoral stages and carefully characterise their functional and molecular properties (Calvo et al., 2013).
Materials and Reagents
FVB/n MMTV-PyMT females between 4 and 14 weeks of age and MMTV-PyMT negative siblings for isolation of normal mammary gland fibroblasts (NFs) (The Jackson Laboratory, catalog number: 00 2374 )
Phoenix-Eco packing cells (ATCC, catalog number: CRL-3214 ) (read Note 6 for more information)
pBabe-HPV-E6-puromycin plasmid (or immortalization plasmid of choice, see Note 6)
Note: This was generated in our laboratory but there are alternatives available (pLenti-puro-HPV-16 E6/E7; Applied Biological Materials, catalog number: G268 ).
Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose with stable L-glutamine) (Life Technologies, Gibco®, catalog number: 41966-029 )
50x sodium butyrate (500 mM)
Polybrene® (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide) (Sigma-Aldrich, catalog number: AL-118 ) (1000x solution at 2 mg/ml)
Insulin-Transferrin-Selenium (ITS) solution (Life Technologies, catalog number: 51300-044 ).
Calcium phosphate transfection kit (ProFection® Mammalian Transfection System) (Promega Corporation, catalog number: E1200 )
Fetal Bovine Serum (FBS)
Phosphate-buffered saline (PBS)
PBS without Ca2+ and Mg2+
Penicillin/Streptomycin
EDTA (1 mM in PBS without Ca2+ and Mg2+)
10% formalin solution, neutral-buffered (10% NBF)
100x Collagenase/dispase (100 mg/ml) (Roche Diagnostics, catalog number: 11097113001 )
1000x puromycin (2 mg/ml) (Sigma-Aldrich, catalog number: P9620 )
1000x DNase I (10 mg/ml) (Sigma-Aldrich, catalog number: D4513 )
Trypsin/EDTA
Virkon (DuPont Rely+On Virkon, catalog number: 1235-8667 )
Complete media (see Recipes)
Equipment
0.22 µm, 0.45 µm, 3 mm filters
20 mm coverslip
Tissue culture plastic-ware
37 °C shaker
Tissue culture laminar-flow hood
10 cm tissue culture dish
Centrifuge
37 °C, 5% CO2, cell culture incubator
Scalpels
Surgical scissors
Dissecting instruments
Procedure
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How to cite：Calvo, F., Hooper, S. and Sahai, E. (2014). Isolation and Immortalization of Fibroblasts from Different Tumoral Stages. Bio-protocol 4(7): e1097. DOI: 10.21769/BioProtoc.1097.
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Category
Cancer Biology > General technique > Animal models
Cancer Biology > Invasion & metastasis > Tumor microenvironment
Cell Biology > Cell isolation and culture > Cell isolation
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1,098 | https://bio-protocol.org/exchange/protocoldetail?id=1098&type=0 | # Bio-Protocol Content
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Quantification of Anthocyanin Content
MN Masaru Nakata
Masaru Ohme-Takagi
Published: Vol 4, Iss 7, Apr 5, 2014
DOI: 10.21769/BioProtoc.1098 Views: 28156
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in May 2013
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Abstract
Anthocyanins are a class of flavonoids and important plant pigments. They attract insects to pollinate flowers, protect plants from UV irradiation, and act as antimicrobial agents against herbivores and pathogens. Biosynthesis of anthocyanin is stimulated by diverse developmental signals and environmental stresses including drought, wounding, pathogen infection and insect attack. Plant hormones such as jasmonates, a stress-related plant hormone, also induce accumulation of anthocyanins. Sensitivity of plants to these stress stimuli can be measured by accumulation of anthocyanins. Here we describe a simple method for measurement of anthocyanins in Arabidopsis thaliana seedlings. Amount of anthocyanins are calculated only from absorbances at 530 and 657 nm of crude extract.
Materials and Reagents
Arabidopsis thaliana seedlings (~10 days after germination)
Note: Amount of anthocyanin per seedling weight is higher in young seedlings.
Bleach solution
Sterile dH2O
Methanol
Acetic acid
Murashige and Skoog medium salt (Wako Pure Chemical Industries, catalog number: 392-00591 )
Sucrose
2-Morpholinoethanesulfonic acid (MES)
Agar (for plant culture)
Modified Murashige and Skoog medium (see Recipes)
Extraction buffer (see Recipes)
Equipment
Paper towel
Spectrophotometer
Microcentrifuge
Microcentrifuge tubes
Mortar and pestle
Liquid nitrogen
Electric balance
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Nakata, M. and Ohme-Takagi, M. (2014). Quantification of Anthocyanin Content. Bio-protocol 4(7): e1098. DOI: 10.21769/BioProtoc.1098.
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Category
Plant Science > Plant biochemistry > Other compound
Biochemistry > Other compound > Flavonoid
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1,099 | https://bio-protocol.org/exchange/protocoldetail?id=1099&type=0 | # Bio-Protocol Content
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Electrophoresis Mobility Shift Assay
MN Masaru Nakata
Masaru Ohme-Takagi
Published: Vol 4, Iss 7, Apr 5, 2014
DOI: 10.21769/BioProtoc.1099 Views: 17243
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in May 2013
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Abstract
Protein (transcription factors and/or transcription cofactors)-binding to DNA is a critical event in regulation of transcription. Electrophoresis Mobility Shift Assay (EMSA), also known as gel shift assay, is a useful tool to detect protein- or protein complex-DNA/RNA interaction and to evaluate DNA binding specificity of transcription factors in vitro. Here we describe a simple method for EMSA with fluorescent dye-bound oligo DNA probes and recombinant protein expressed in bacterial cells. Using fluorescent dye instead of radioisotope enables easy handling and long-term storage of labelled-probes without reduction of detection sensitivity.
Materials and Reagents
Oligo DNA 5’ end-labeled with IRDye 700 or IRDye 800 (sense strand) (Integrated DNA Technologies)
Non-labelled oligo DNA (both sense and antisense strands)
Non-labelled mutated oligo DNA (both sense and antisense strands)
Recombinant DNA-binding proteins expressed in Escherichia coli (E. coli) (10 ng/µl in protein storage buffer)
Sterile distilled water (SDW)
Odyssey infrared EMSA kit (LI-COR, catalog number: 829-07910 )
Poly(dI-dC) (Sigma-Aldrich, catalog number: 4929 )
Tris
Boric acid
EDTA 2Na
NaCl
HCl
Acrylamide
N,N’-Methylene-bisacrylamide
Glycerol
Triton X-100
Phenylmethylsulfonyl fluoride (PMSF)
β-mercaptoethanol
Ammonium persulfate (APS)
N,N,N',N'-tetramethylethylenediamine (TEMED)
10x TBE( see Recipes)
4% native polyacrylamide gel (see Recipes)
Native-PAGE running buffer (see Recipes)
Protein storage buffer (see Recipes)
Equipment
Odyssey CLx Infrared Imaging System (LI-COR)
A set of devices for polyacrylamide gel electrophoresis
Power supply
Refrigerator or cold room
Heat block
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Nakata, M. and Ohme-Takagi, M. (2014). Electrophoresis Mobility Shift Assay. Bio-protocol 4(7): e1099. DOI: 10.21769/BioProtoc.1099.
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Category
Molecular Biology > DNA > DNA-protein interaction
Biochemistry > Protein > Interaction
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11 | https://bio-protocol.org/exchange/protocoldetail?id=11&type=1 | # Bio-Protocol Content
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How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells
Bio-protocol Editor
Published: Jan 5, 2011
DOI: 10.21769/BioProtoc.11 Views: 23347
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Abstract
The EmulsiFlex-C3 homogenizer is powered by an electric motor. The pump does not require a compressor for it to run. This equipment can be used to disrupt cells at a large scale. The EmulsiFlex-C3 has a fixed flow-through capacity of 3 L/h. It has the ability to process samples as small as 10 ml. The homogenizing pressure is adjustable between 500 and 30,000 psi. In this protocol, we describe the use of the Avestin Emulsiflex C3 Homogenizer to disrupt S. pombe and S. cerevisiae cells.
Materials and Reagents
S. pombe cells
S. cerevisiae cells
DI water
Equipment
Avestin Emulsiflex C3 homogenizer (Avestin®)
Figure 1. Avestin Emulsiflex C3 homogenizer
Standard laboratory bench-top light microscope
Procedure
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cell Biology > Cell viability > Cell lysis
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1,100 | https://bio-protocol.org/exchange/protocoldetail?id=1100&type=0 | # Bio-Protocol Content
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Zonal Sedimentation Analysis on Sucrose Gradients
Javier G. Magadán
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1100 Views: 9559
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The authors used this protocol in Sep 2013
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Abstract
Zonal sedimentation analysis on sucrose gradients allows estimation of the molecular size of an individual protein or a protein complex by centrifugation at a constant speed under nondenaturing conditions. This method is particularly suitable for globular proteins like the influenza A virus (IAV) protein hemagglutinin (HA). Here, I describe step by step a protocol used to evaluate the oligomeric state of recombinant HA trimers (Magadan et al., 2013).
Materials and Reagents
Trimerized recombinant HA (recHA3) derived from influenza A/Puerto Rico/8/34 (PR8) virus (Magadan et al., 2013)
Gel filtration protein standards [carbonic anhydrase (29 kDa), ovalbumin (43 kDa), conalbumin (75 kDa), aldolase (158 kDa), and ferritin (440 kDa)] (GE, catalog numbers: 28-4038-41 and 28-4038-42 )
4x NuPAGE LDS sample buffer (Life Technologies, catalog number: NP0007 )
NuPAGE Novex 4-12% Bis-Tris protein gels (Life Technologies, catalog number: NP0321PK2 )
NuPAGE MES SDS running buffer (Life Technologies, catalog number: NP000202 )
Ponceau S solution (Sigma-Aldrich, catalog number: P7170 )
5% acetic acid
Blotting grade blocker nonfat dry milk (Bio-Rad Laboratories, catalog number: 170-6404XTU )
1x PBS (Life Technologies, catalog number: AM9624 )
Tween-20 (Sigma-Aldrich, catalog number: P1379 )
A home-made, conformation-independent mouse monoclonal antibody to denatured HA1 (clone CM-1)
A rabbit polyclonal anti-mouse HRP-conjugated antibody (Dako, catalog number: P0260 )
SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34077 )
High purity sucrose (Thermo Fisher Scientific, catalog number: NC0110097 )
UltraPure 5 M NaCl (Life Technologies, catalog number: 24740-011 )
UltraPure 0.5 M EDTA (pH 8.0) (Life Technologies, catalog number: 15575-020 )
Sucrose gradient (see Recipes)
Equipment
Refractometer (Bausch & Lomb Incorporated)
14 x 89 mm Ultra Clear tubes (Beckman Coulter, catalog number: 344059 )
Pipettor
1.5 ml micro-centrifuge tubes
An ultracentrifuge equipped with a SW41 rotor (Beckman Coulter)
Nitrocellulose blotting membranes (0.45 µm pore size) (Life Technologies, catalog number: LC2000 )
A chamber to run mini-gels [I routinely use the XCell SureLock Mini Cell electrophoresis system (Life Technologies, catalog number: EI0001 ).]
A Mini Trans-Blot Cell (Bio-Rad Laboratories, catalog number: 170-3930 )
Carestream Kodak BioMax XAR films (Sigma-Aldrich, catalog number: F5388 )
Kodak X-OMAT 2000A processor or equivalent
A cassette for autoradiography
Software
ImageJ software (http://imagej.nih.gov/ij/)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Magadán, J. G. (2014). Zonal Sedimentation Analysis on Sucrose Gradients. Bio-protocol 4(8): e1100. DOI: 10.21769/BioProtoc.1100.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Structure
Biochemistry > Protein > Electrophoresis
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1,101 | https://bio-protocol.org/exchange/protocoldetail?id=1101&type=0 | # Bio-Protocol Content
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Radioactive Pulse-Chase Analysis and Immunoprecipitation
Javier G. Magadán
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1101 Views: 12187
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The authors used this protocol in Sep 2013
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Abstract
Labeling of newly-synthesized polypeptides with radioactive amino acids followed by immunoprecipitation allows quantitative analysis of the fate of a given protein in a time-dependent manner. This biochemical approach is usually used to study a variety of processes, such as protein folding, co-translational modifications, intracellular transport, and even its rate of degradation. Here, I describe step by step a simple technique to both label newly-synthesized influenza A virus (IAV) hemagglutinin (HA) with [35S]-methionine and then follow its maturation and transport through the secretory pathway by SDS-PAGE and fluorography (Magadan et al., 2013).
Materials and Reagents
~5 x 106 MDCK cells (ATCC, catalog number: CCL-34 )
0.05% 1x Trypsin-EDTA (phenol red) (Life Technologies, catalog number: 25300054 )
DPBS (Life Technologies, catalog number: 14190-250 )
Anti-HA antibodies (mostly home-made)
nProtein A Sepharose 4 Fast Flow (GE, catalog number: 17-5280-02 )
4x NuPAGE LDS sample buffer (Life Technologies, catalog number: NP0007 )
NuPAGE Novex 4-12% Bis-Tris protein gels (Life Technologies, catalog number: NP0321PK2 )
NuPAGE MES SDS running buffer (Life Technologies, catalog number: NP000202 )
DMEM media without methionine and cysteine (Life Technologies, catalog number: 21013024 )
EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693159001 )
10% Triton X-100 surfact-amps detergent solution (Thermo Fisher Scientific, catalog number: 28314 )
UltraPure 1 M Tris-HCI Buffer (pH 7.5) (Life Technologies, catalog number: 15567-027 )
UltraPure 5 M NaCl (Life Technologies, catalog number: 24740-011 )
UltraPure 0.5 M EDTA (pH 8.0) (Life Technologies, catalog number: 15575-020 )
Methanol (Sigma-Aldrich, catalog number: 322415 )
Acetic acid (Sigma-Aldrich, catalog number: 320099 )
DMEM media (Life Technologies, catalog number: 10569-010 )
DMEM media with 7.5% fetal bovine serum (see Recipes)
Pulse medium (see Recipes)
Chase medium (see Recipes)
Non-denaturing lysis buffer (see Recipes)
Fixation solution (see Recipes)
L-cysteine (Sigma-Aldrich, catalog number: W326305 ) (see Recipes)
[35S]-methionine (PerkinElmer, catalog number: NEG-709-A005MC ) (see Recipes)
L-methionine (Sigma-Aldrich, catalog number: M9625 ) (see Recipes)
Equipment
Note: Designated for working with radioactive materials.
Appropriate containers to dispose liquid and solid [35S]-waste following local radiation safety guidelines
T-75 tissue culture flask (Thermo Fisher Scientific)
50 ml BD Falcon tube (BD Biosciences, Falcon®)
A water bath set at 37 °C
A refrigerated micro-centrifuge
A rotator for 1.5 ml micro-centrifuge tubes settled at 4 °C
1.5 ml micro-centrifuge tubes
A chamber to run mini-gels [I routinely use the XCell SureLock Mini Cell electrophoresis system (Life Technologies, catalog number: EI0001 ).]
Carestream Kodak BioMax MR films (Sigma-Aldrich, catalog number: Z350400 )
Kodak X-OMAT 2000A processor
A gel-dryer
A cassette for autoradiography
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Magadán, J. G. (2014). Radioactive Pulse-Chase Analysis and Immunoprecipitation. Bio-protocol 4(8): e1101. DOI: 10.21769/BioProtoc.1101.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Immunodetection
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1,102 | https://bio-protocol.org/exchange/protocoldetail?id=1102&type=0 | # Bio-Protocol Content
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Protocol for the Preparation of Arabidopsis Meiotic Chromosome Spreads and Fluorescent in situ Hybridization
Pablo Bolaños-Villegas
XY Xiaohui Yang
Christopher A. Makaroff
Guang-Yuh Jauh
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1102 Views: 15226
Edited by: Renate Weizbauer
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
This protocol is a more detailed version of previous protocols (Yang et al., 2011; Bolaños-Villegas et al., 2013) developed for the examination of meiotic chromosome spreads. Meiotic chromosome spreads are useful to determine the presence of defects in chromosome pairing and segregation. The protocol also describes how to perform fluorescent in situ hybridization experiments with a centromere probe used to label chromosomes.
Keywords: Meiocyte Cell cycle Centromere Reproduction Higher plants
Materials and Reagents
Inflorescences from young, healthy Arabidopsis thaliana plants
Agar plates, with 1/2x Murashige and Skoog solid medium
Pectolyase (Sigma-Aldrich, catalog number: P3026 )
Cellulase (Sigma-Aldrich, catalog number: C1184 )
β-glucoronidase (Roche Diagnostics, catalog number: 03707598001 )
Sucrose (Affymetrix, catalog number: 21938 )
Ethanol (EMD Millipore, catalog number: 1085430250 )
Chloroform (EMD Millipore, catalog number: 102442 )
Glacial acetic acid (EMD Millipore, catalog number: 100063 )
Citric acid anhydrous (Affymetrix, catalog number: 13729 )
Sodium citrate dihydrate (Affymetrix, catalog number: 13735 )
Formamide (Sigma-Aldrich, catalog number: F9037 )
Saline-sodium citrate (SSC) (Sigma-Aldrich, catalog number: S0902 )
Dextran sulfate (Sigma-Aldrich, catalog number: 42867 )
Salmon sperm (Life Technologies, catalog number: 15632-011 )
VECTASHIELD® mounting medium with DAPI (10 ml) (Vector Laboratories, catalog number: H-1400 )
Fluorescein High-Prime labeling kit (Roche Diagnostics, catalog number: 11585622910 )
pAL1 centromere probe (ABRC, catalog number: CD3-16 )
Taq DNA polymerase with standard Taq buffer (New England Biolabs, catalog number: M0273S )
QIAquick Gel Extraction Kit (kit for purification of DNA from gels) (QIAGEN, catalog number: 28704 )
Gel Pilot 1 Kb Ladder (molecular weight marker for DNA) (QIAGEN, catalog number: 239085 )
Agarose for electrophoresis (Bio-Rad Laboratories, catalog number: BR 161-3100 )
Carnoy’s solution (see Recipes)
10 mM Sodium citrate buffer (see Recipes)
Enzyme digestion buffer (see Recipes)
FISH buffer (see Recipes)
10x PBS (see Recipes)
20x SSC (see Recipes)
Equipment
Hypodermic needles and syringe (Terumo Medical Corporation, model: 26G x ½”, catalog number: NN-2613R )
Tweezers (Dumont)
Poly-Prep® poly-L-lysine coated slides (Sigma-Aldrich, catalog number: P0425-72EA )
Nunc® Lab-Tek® II slides (Thermo Fisher Scientific, catalog number: 154453 )
Coverslips (Matsunami Glass, catalog number: C218181 )
Hybridisation oven OV1 (Biometra, catalog number: 052-090 )
Thermal cycler (Biometra, catalog number: 050-551 )
Dry block (Thermo Fisher Scientific, Reacti-Therm®)
-86 °C refrigerator (Thermo Fisher Scientific, model: Forma® 88000 )
Frigidaire -20 °C refrigerator (Frigidaire, model: FCFS201LFB )
Fluorescence microscope
Coplin staining jars (Thermo Fisher Scientific, catalog number: 107 )
Rubbermaid LunchBlox® food containers
Super Pap Pen’ liquid blocker pen (Electron Microscopy Sciences, catalog number: 71310 ) (optional)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell structure > Chromosome
Molecular Biology > DNA > DNA structure
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1,103 | https://bio-protocol.org/exchange/protocoldetail?id=1103&type=0 | # Bio-Protocol Content
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Purification and Fluorescent Labeling of Exosomes
AN Asuka Nanbo
EK Eri Kawanishi
RY Ryuji Yoshida
HY Hironori Yoshiyama
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1103 Views: 19884
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
Exosomes are small membrane vesicles of endocytic origin secreted into the extracellular environment from a variety of different cells, and are thought to play important roles in intercellular communications. Here, we provide a useful protocol to purify the exosomes released from cell lines using sucrose gradient centrifugation. In this protocol, we also applied a red-fluorescent lipophilic dye, DiI, which is incorporated in the outer membrane of exosomes. This fluorescently labeled exosomes allow us to visualize individual exosomes by a confocal laser scanning microscope.
Keywords: Exosome Extracellular vesicle Fluorescent labeling Microscope
Materials and Reagents
Burkitt’s Lymphoma B cell lines (e.g. Mutu-, Mutu I, Mutu III cell lines)
RPMI 1640 medium (Wako Chemicals USA, catalog number: 189-02025 )
Sucrose (Sigma-Aldrich, catalog number: S7903 )
Anti-CD63 monoclonal antibody (clone MEM-250) (Abnova, catalog number: MAB0931 )
Bradford protein assay kit (Bio-Rad Laboratories, catalog number: 500-0006JA )
1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) (Life Technologies, catalog number: D3911 )
Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F9423 )
Tris
NaCl
EDTA
Exosome-depleted FBS (see Recipes)
TNE buffer (see Recipes)
0.25-2.5 M sucrose gradient in TNE buffer (see Recipes)
Equipment
10 cm dish
Centrifuge (Eppendorf, model: 5810R or that with equivalent equipment spec)
Ultracentrifuge (Beckman Coulter, model: Optima L-80 XP or that with equivalent equipment spec)
37 °C, 5% CO2 cell culture incubator
Autopipette
50 ml polypropylene concal plastic tubes (BD Biosciences, Falcon®, catalog number: 352070 or that with equivalent spec)
SW28 rotor (Beckman Coulter, model: 342204 )
SW40Ti rotor (Beckman Coulter, model: 331301 )
Polyallomer centrifuge tubes 1 x 3½ in (25 x 89 mm) for SW28 rotor (Beckman Coulter, catalog number: 326823 )
Polyallomer centrifuge tubes 9/16 x 3½ in (14 x 89 mm) for SW41Ti rotor (Beckman Coulter, catalog number: 331372 )
Spectrometer
Fluorescent or confocal laser scanning microscope
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Nanbo, A., Kawanishi, E., Yoshida, R. and Yoshiyama, H. (2014). Purification and Fluorescent Labeling of Exosomes. Bio-protocol 4(8): e1103. DOI: 10.21769/BioProtoc.1103.
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Category
Cell Biology > Organelle isolation > Exosomes
Cell Biology > Cell imaging > Fluorescence
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1,104 | https://bio-protocol.org/exchange/protocoldetail?id=1104&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vivo Lineage-tracing Studies in a Cancer Stem Cell Population in Neuroblastoma
DH Danielle M. Hsu
JS Jason M. Shohet
Eugene S. Kim
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1104 Views: 9357
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Jul 2013
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Abstract
Tumors are comprised of heterogeneous subpopulations that may exhibit differing capacity for differentiation, self-renewal, and tumorigenicity. In vivo lineage-tracing studies are a powerful tool for defining the role of tumor subpopulations in tumor growth and as targets for therapeutic agents. This protocol describes using a neuroblastoma cancer cell line transduced with two different fluorescent proteins (GFP and tdTomato) to track the specific contributions of cells expressing the GCSF receptor (CD114+) or not (CD114-) on tumor growth in vivo.
Materials and Reagents
Human neuroblastoma cell lines (NGP, NB-1691, IMR-32)
Note: A cell line is transduced with two different fluorescent proteins, for example GFP (Clontech, catalog number: 632370 ) and tdTomato (Clontech, catalog number: 632534 ), such that there is a GFP positive line and a tdTomato line of the same cell type. In this manner, subpopulations of the same cell type (i.e. GCSF-R positive and GCSF-R negative cells) can be traced. If in vivo monitoring of tumor growth via bioluminescent imaging is desired, cell lines should also be transfected or virally transduced with commercially available vectors, e.g. pGL2-Control Vector (Promega Corporation) to express a luminescent reporter gene. For a detailed description and protocol of in vivo bioluminescent imaging, please refer to Reference 2.
4-6 week old female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Taconic, model number: NODSCF , http://www.taconic.com/NODSC)
293T cells
RPMI medium 1640 (Life Technologies, catalog number: 11875-101 )
10% (v/v) fetal bovine serum (FBS) (Life Technologies, catalog number: 16000044 )
1% (v/v) penicillin/streptomycin (Life Technologies, catalog number: 15140122 ) (100x P/S; final concentration 100 units/ml penicillin and 100 mg/ml streptomycin)
1% (v/v) of 100x L-glutamine (Life Technologies, catalog number: 25030081 ) (final concentration 2 mM)
Phosphate-buffered saline (PBS) (sterile) (Life Technologies, catalog number: 70011069 )
0.25% trypsin/EDTA (Life Technologies, catalog number: 25200056 )
Collagenase I (Sigma-Aldrich, catalog number: C0130 ) [prepare a solution containing 10,000 Collagenase Digestion Unites (CDU/ml) in PBS]
Dispase II (Roche Diagnostics, catalog number: 04942078001 ) (prepare a solution containing 32 mg/ml Dispase II in PBS)
DNase I (EMD Millipore, Calbiochem®, catalog number: 260913 ) (prepare a solution containing 5 MU/ml DNase I)
FuGENE 6 (Promega Corporation, catalog number: E2691 )
Opti-MEM Reduced Serum Medium (Life Technologies, catalog number: 31985-062 )
PE conjugated anti-CD 114 (GCSFR) antibody (BD Biosciences, catalog number 554538 )
Cell culture medium (see Recipes)
Sterile FACS buffer (see Recipes)
PEB Buffer (see Recipes)
Equipment
Fluorescence-activated cell sorter (e.g. DAKO Cytomation MoFlo 9-color cell sorter)
37 °C, 5% CO2 tissue culture incubator
Refrigerated centrifuge
Class 2 biological safety cabinet with laminar flow hood
70 μm cell strainer (Thermo Fisher Scientific, catalog number: 22-363-548 )
T-75 culture flask or 10 cm dish
Anesthesia machine/chamber with nose cone appropriate for mice (Surgivet or VetEquip)
Fluorescent microscope
Surgical instruments
5.5-in Mayo-Hegar or similar surgical needle holder (Millennium Surgical or Roboz Surgical Instrument)
Sterile gloves (Thermo Fisher Scientific)
Disposable sterile scalpel blade (#10) (Millennium Surgical or Roboz Surgical Instrument)
27-G needle
Sterile 1-cc slip tip syringe
Polysorb 4-0 sutures with RB-1 tapered needle (U.S. Surgical)
9-mm wound clips (VWR International)
Rodent ear tags (National Band & Tag Company)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cancer Biology > Invasion & metastasis > Tumor microenvironment
Cancer Biology > Proliferative signaling > Tumor formation
Stem Cell > Adult stem cell > Cancer stem cell
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1,105 | https://bio-protocol.org/exchange/protocoldetail?id=1105&type=0 | # Bio-Protocol Content
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Peer-reviewed
Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines
Amit K. Dey
AB Abha Bhagat
RC Rukhsana Chowdhury
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1105 Views: 10690
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in May 2013
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May 2013
Abstract
Vibrio cholerae (V. cholerae) colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes severe fluid loss characteristic of the disease cholera. V. cholerae is a non-invasive Gram-negative bacterium that adheres to intestinal cells as well as a variety of different cell types. A protocol for adherence of V. cholerae to various cell lines is described. The adhered bacteria can be used to examine expression of genes that are differentially expressed between adhered and unadhered bacteria or other purposes (Dey et al., 2013).
Keywords: Adherence to INT407 Vibrio cholerae adherence Host pathogen interaction Virulence gene induction VieA induction
Materials and Reagents
Cell lines: INT 407, Hep-2, Hela, HT-29 (available from ATCC and other commercial sources)
Vibrio cholerae classical biotype strain O395
Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco®)
High glucose (4,500 mg/L) (catalog number: 12800-017 )
Low glucose (1,000 mg/L) (catalog number: 31600-034 )
No glucose (catalog number: 11966-025 )
New born calf serum (NCS) (not heat inactivated) (Life Technologies, Gibco®, catalog number: 16010-159 )
10x Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 15400 )
TritonX-100 (Affymetrix, catalog number: T1001 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: C6164 )
D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021 )
D-Galactose
10x cell lysis buffer (Cell Signaling Technology, catalog number: 9803 )
pNPP (5 mg) (Sigma Chemical, catalog number: N-9389 )
Bacto tryptone (BD, DifcoTM, catalog number: 211705 )
Bacto yeast extract (BD, DifcoTM, catalog number: 212750 )
Bacto agar (BD, DifcoTM, catalog number: 214010 )
NaHCO3 (Merck KGaA)
NaCl (Merck KGaA)
Na2HPO4 (Merck KGaA)
KCl (Merck KGaA)
KH2PO4 (Merck KGaA)
70% ethanol (Merck KGaA)
NaOH (Merck KGaA)
Personal protective items (apron, gloves, mask etc.)
Bio-Rad protein assay dye reagent (Bio-Rad Laboratories)
Complete DMEM (100 ml) (see Recipes)
Incomplete DMEM (100 ml) (see Recipes)
No glucose complete DMEM (100ml) (see Recipes)
10x PBS (500 ml, pH 7.4) (see Recipes)
1x PBS (50 ml) (see Recipes)
1x Trypsin-EDTA (10 ml) (see Recipes)
1% Triton X-100 (50 ml) (see Recipes)
0.9% saline solution (see Recipes)
LB medium (100 ml) (see Recipes)
LB Agar (100 ml) (see Recipes)
Alkaline phosphatase assay buffer (10 ml) (see Recipes)
Equipment
25 cm2 tissue-culture treated flasks (BD Biosciences, Falcon®, catalog number: 353108 )
Sterile, disposable 15 ml and 50 ml centrifuge tubes
Sterile, disposable 1.5 ml microcentrifuge tubes
Sterile microtips
Sterile cryovials
Cell scraper (BD Biosciences, Falcon®)
0.22 μm filter units (Millex-GP) (Millipore, catalog number: SLGP033RS )
CO2 incubator
Laminar flow hood
Liquid nitrogen storage container
Inverted microscope
Centrifuge
Microcentrifuge
Pipetteman
UV-Vis Spectrophotometer
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Dey, A. K., Bhagat, A. and Chowdhury, R. (2014). Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines. Bio-protocol 4(8): e1105. DOI: 10.21769/BioProtoc.1105.
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Category
Microbiology > Microbe-host interactions > In vitro model
Cell Biology > Cell structure > Cell adhesion
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1,106 | https://bio-protocol.org/exchange/protocoldetail?id=1106&type=0 | # Bio-Protocol Content
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Peer-reviewed
Sandwich Enzyme-linked Immunosorbent Assay (ELISA) Analysis of Plant Cell Wall Glycan Connections
Valérie Cornuault
JK J. Paul Knox
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1106 Views: 16600
Edited by: Renate Weizbauer
Original Research Article:
The authors used this protocol in Sep 2013
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Sep 2013
Abstract
Sandwich ELISA is a highly sensitive method that can be used to determine if two epitopes are part of the same macromolecule or supramolecular complex. In the case of plant cell wall glycans, it can reveal the existence of inter-polymers linkages, leading to better understanding of overall cell wall architectures. This development of a conventional sandwich ELISA protocol uses a carbohydrate-binding module (CBM), a small protein domain found in some carbohydrate catalysing or activating enzymes, and rat monoclonal antibodies (mAbs) which can be combined in the same ELISA plate without risk of cross reaction; the secondary anti-rat HRP antibody being only able to bind to the rat mAb and not the CBM. This protocol was developed and modified in the Prof. J. Paul Knox lab at the University of Leeds.
Figure 1. Sandwich ELISA analysis of complex glycans
Materials and Reagents
Cell wall extracts [50 mM cyclohexanediamine tetraacetic acid (CDTA) (Sigma-Aldrich, catalog number: D1383 ) or 4 M KOH/1% (w/v) NaBH4 (Sigma-Aldrich, catalog number: 452882 ) or purified cell wall polymers]
Notes:
Reagents for cell wall extracts are 50 mM cyclohexanediamine tetraacetic acid (CDTA) (Sigma-Aldrich, catalog number: D1383) or 4 M KOH/1% (w/v) NaBH4 (Sigma-Aldrich, catalog number: 452882)
Cell wall extraction protocol using 4 M KOH/1% (w/v) NaBH4 is described in Cid et al. (2010). This extraction will disrupt and release most cell wall glycans.
CDTA is a chelating agent which is used to specifically extract the pectin fraction from cell walls. The same protocol can be followed using 50 mM CDTA instead of 4 M KOH (no neutralizing step necessary).
Skimmed milk powder (Marvel Original) [used at 5% (w/v) in 1x PBS]
Purified recombinant carbohydrate-binding module (CBM)
Note: A protocol for CBM expression and purification can be found in Lee et al. (2013).
Primary monoclonal antibody (PlantProbes, www.plantprobes.net)
Secondary antibody (depending on the origin of the primary antibody used)
Anti-rat IgG-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich, catalog number: A9542 )
Anti-mouse IgG-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich, catalog number: A6782 )
10 mg/ml tetramethyl benzidine (Sigma-Aldrich, catalog number: T2885 )
1 M sodium acetate buffer (pH 6)
6% H2O2 (VWR International, catalog number: 2858175C )
2.5 M H2SO4 (VWR International, catalog number: 191675A )
Tween 20 (Sigma-Aldrich, catalog number: P2287 ) (optional)
1x phosphate-buffered saline (PBS) (pH 7) (see Recipes)
HRP developing solution (see Recipes)
Equipment
96-well surface treated ELISA microtitre plates (Thermo Fisher Scientific, Nunc-Immnuno Maxisorp, catalog number: 442404 )
Microtitre plate reader (450 nm absorbance) (Thermo Fisher Scientific, MultiskanTM FC Microplate Photometer)
Multi-channel pipette (if available, not critical)
pH meter
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Cornuault, V. and Knox, J. P. (2014). Sandwich Enzyme-linked Immunosorbent Assay (ELISA) Analysis of Plant Cell Wall Glycan Connections. Bio-protocol 4(8): e1106. DOI: 10.21769/BioProtoc.1106.
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Category
Plant Science > Plant biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Polysaccharide
Biochemistry > Protein > Immunodetection
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1,107 | https://bio-protocol.org/exchange/protocoldetail?id=1107&type=0 | # Bio-Protocol Content
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Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP
CR Christian Roth
JG Janosch Alexander D. Gröning
SK Stefan Rudolf Kaschabek
MS Michael Schlömann
N Norbert Sträter
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1107 Views: 10546
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Apr 2013
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Abstract
The protocol describes the generation of variants of chloromuconolactone dehalogenase from Rhodococcus opacus (R. opacus) 1CP. ClcF is a multimeric protein, which catalyses the dechlorination of 5-chloromuconolactone to cis-dienelactone in the 3-chlorocatecholic acid degradation pathway. The protocol describes the workflow for the purification and subsequent crystallization of the enzyme. The used workflow and the described techniques could be easily adapted to any other protein/enzyme intended to be crystallized by the potential user for subsequent structure determination. The protocol does not involve expensive specialized equipment which allows the use in standard laboratories not specially dedicated to macromolecular crystallography.
Keywords: Crystallisation Protein purification Column chromatography Recombinant protein expression Biochemistry
Materials and Reagents
Escherichia coli (E. coli) BL21 (DE3)-CP-RIL (Stratagene, catalog number: 230245 )
E.coli DH5α (Life Technologies, catalog number: 18258012 )
wtClcF plasmid (not commerically available)
Forward Primer (not commerically available)
Reverse Primer (not commerically available)
dNTP-Mix (20 mM) (Thermo Fisher Scientific, catalog number: AB-0196
Pfu-Ultra (2.5 U/µl) (Agilent, catalog number: 600385 )
DpnI (Thermo Fisher Scientific, catalog number: ER1701 )
LB medium
Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Carl Roth, catalog number: CN08.1 )
Tris base (Carl Roth, catalog number: 5429.1 )
DNase (Sigma-Aldrich, catalog number: DN25-100MG )
Ethylene glycol
MgCl2 (Carl Roth, catalog number: 2189.2 )
PEG 3350 (Sigma-Aldrich, catalog number: 202440-250G )
Bis-Tris (Carl Roth, catalog number: 9140.1 )
Liquid nitrogen
Lysis buffer/Ion exchange (IEX) buffer A/Hydrophobic interaction chromatography (HIC) buffer A (see Recipes)
HIC High salt buffer (see Recipes)
Wash buffer (see Recipes)
Crystallization buffers (see Recipes)
Equipment
Standard laboratory equipment
Nanodrop device (Thermo Fisher Scientific)
CrystalQuickTM 96 well crystallization plate Greiner 609101(Jena Bioscience, catalog number: CPL-118S )
Linbro crystallization plate (Jena Bioscience, catalog number: CPL-101S )
22 mm circular cover slides (siliconized) (Jena Bioscience, catalog number: CSL-106 )
Bayer silicone grease (Jena Bioscience, catalog number: CGR-101 )
Äkta purification system (GE Healthcare)
HiTrap Q sepharose column (1 ml) (GE Healthcare, catalog number: 17-5053-01 )
HiTrap phenyl-sepharose (1 ml) (GE Healthcare, catalog number: 17-1351-01 )
Stereo microscope (Leica Microsystems, model: MZ12 )
Eppendorf tubes
Pipetting robot (if available)
37 °C incubator
Centrifuge
Vivaspin concentrator (MWCO 30 kDa) (EMD Millipore)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Roth, C., Gröning, J. A. D., Kaschabek, S. R., Schlömann, M. and Sträter, N. (2014). Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP. Bio-protocol 4(8): e1107. DOI: 10.21769/BioProtoc.1107.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Structure
Biochemistry > Protein > Isolation and purification
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1,108 | https://bio-protocol.org/exchange/protocoldetail?id=1108&type=0 | # Bio-Protocol Content
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Histochemical Detection of Superoxide and H2O2 Accumulation in Brassica juncea Seedlings
Deepak Kumar
Mohd Aslam Yusuf
Preeti Singh
Meryam Sardar
Neera Bhalla Sarin
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1108 Views: 32995
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Oct 2013
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Oct 2013
Abstract
Plant cells continually produce reactive oxygen species (ROS) as a by-product of aerobic metabolism. Increased production of ROS occurs under unfavorable conditions imposed by various abiotic and biotic factors. Accumulation of ROS is damaging to various cellular components and macromolecules including plasma membrane, nucleic acids, and proteins and eventually leads to cell death. In this protocol, we describe the histochemical detection of hydrogen peroxide (H2O2) and superoxide (O2-) anion, two of the most important ROS, in Brassica juncea seedlings by using 3,3ʹ-Diaminobenzidine (DAB) and Nitrotetrazolium blue chloride (NBT) as the chromogenic substrate. DAB is oxidized by H2O2 in the presence of peroxidases and produces reddish brown precipitate. NBT reacts with O2- to form a dark blue insoluble formazan compound. The protocol can be used in other plant species and for different plant tissues.
Materials and Reagents
Brassica juncea seedlings
Vermiculite
Absolute ethanol
60% Glycerol
Aluminium foil
Test tubes
Measuring cylinder
Distilled water
Nitrotetrazolium blue chloride (NBT) (Sigma-Aldrich, catalog number: N6639 )
3,3ʹ-Diaminobenzidine (DAB) (Sigma-Aldrich, catalog number: D8001 )
Sodium phosphate buffer (see Recipes)
NBT staining solution (see Recipes)
DAB staining solution (see Recipes)
Equipment
Paper towel
Weighing balance
Aluminium foil
Magnetic stirrer
pH meter
Water bath
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kumar, D., Yusuf, M. A., Singh, P., Sardar, M. and Sarin, N. B. (2014). Histochemical Detection of Superoxide and H2O2 Accumulation in Brassica juncea Seedlings. Bio-protocol 4(8): e1108. DOI: 10.21769/BioProtoc.1108.
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Category
Plant Science > Plant biochemistry > Other compound
Biochemistry > Other compound > Reactive oxygen species
Cell Biology > Cell staining > Reactive oxygen species
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1,109 | https://bio-protocol.org/exchange/protocoldetail?id=1109&type=0 | # Bio-Protocol Content
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Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): Analysis of Flow Data
Andrew W. Sylwester
SH Scott G. Hansen
LP Louis J. Picker
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1109 Views: 15682
Reviewed by: Jia Li
Original Research Article:
The authors used this protocol in Nov 2012
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Original research article
The authors used this protocol in:
Nov 2012
Abstract
What was initially termed ‘CFC’ (Cytokine Flow Cytometry) is now more commonly known as ‘ICS’ (Intra Cellular Staining), or less commonly as ‘ICCS’ (Intra Cellular Cytokine Staining). The key innovations were use of an effective permeant (allowing intracellular staining), and a reagent to disrupt secretion (trapping cytokines, thereby enabling accumulation of detectable intracellular signal). Because not all researchers who use the technique are interested in cytokines, the ‘ICS’ term has gained favor, though ‘CFC’ will be used here.
CFC is a test of cell function, exposing lymphocytes to antigen in culture, then measuring any cytokine responses elicited. Test cultures are processed so as to stain cells with monoclonal antibodies tagged with fluorescent markers, and to chemically fix the cells and decontaminate the samples, using paraformaldehyde.
CFC provides the powers of flow cytometry, which includes bulk sampling and multi-parametric cross-correlation, to the analysis of antigen-specific memory responses. A researcher using CFC is able to phenotypically characterize cells cultured with test antigen, and for phenotypic subsets (e.g. CD4+ or CD8+ T cells) determine the % frequency producing cytokine above background level.
In contrast to ELISPOT and Luminex methods, CFC can correlate production of multiple cytokines from particular, phenotypically-characterized cells. The CFC assay is useful for detecting that an individual has had an antigen exposure (as in population screenings), or for following the emergence and persistence of antigen memories (as in studies of vaccination, infections, or pathogenesis). In addition to quantifying the % frequency of antigen-responding cells, mean fluorescence intensity can be used to assess how much of a cytokine is generated within responding cells.
With the technological advance of flow cytometry, a current user of CFC often has access to 11 fluorescent channels (or even 18), making it possible to either highly-characterize the phenotypes of antigen-responding cells, or else simultaneously quantify the responses according to many cytokines or activation markers. Powerful software like FlowJo (TreeStar) and SPICE (NIAID) can be used to analyse the data, and to do sophisticated multivariate analysis of cytokine responses.
The method described here is customized for cells from Rhesus macaque monkeys, and the extensive annotating notes represent a decade of accumulated technical experience. The same scheme is readily applicable to other mammalian cells (e.g. human or mouse), though the exact antibody clones will differ according to host system. The basic method described here incubates 1 x 106 Lymphocytes in 1 ml tube culture with antigen and co-stimulatory antibodies in the presence of Brefeldin A, prior to staining and fixation.
Note: This is the second part of a two-part procedure. Part one has the same initial title, but the subtitle “From assay set-up to data acquisition (Sylwester et al., 2014)”. The Abstract and Historical Background is the same for both documents.
Keywords: ICS CFC Cytometry Memory PBMC
[Historical Background] In 1988, Andersson, et al. first demonstrated how lymphocytes could be fixed, permeabilized, stained with antibodies against IFNg, then fluorescently labeled and enumerated by flow cytometry. In 1991, Sander et al. demonstrated improved methods to fix cells with paraformaldehyde, permeabilize them with saponin, then use fluorescently-labeled antibodies to stain intracellular cytokines for microscopic examination. In 1993, Jung et al. extended this method to use with flow cytometry, and included the use of monensin to disrupt secretion, so as to increase intracellular signal of molecules otherwise released soon after synthesis. In 1995, Prussin and Metcalfe used directly-conjugated antibodies, and optimized the incubation period to 6 h. Also in 1995, Picker et al. considerably enhanced the sensitivity and reproducibility of cytokine detection by using Brefeldin A to block the secretion apparatus for cytokines, and by using a different permeant (Tween-20). In 1997, this matured method was applied by Picker et al. to study the the antigen-specific homeostatic mechanism in HIV+ patients. In 2001, Schuerwegh et al. confirmed that BfA provides for better cytokine signals than monensin, used by others in this method.
In two reports in 1989, one by Gardner et al. and the other by McClure et al. reported that Rhesus macaques were a useful model for studying HIV disease and AIDS. In 2002, Picker et al. reported the application of the CFC assay to Rhesus macaques. In 2012, a group created to develop multi-lab standards for use of ICS in NHP vaccine studies published their recommendations for a 96-well plate method with a 6 h total incubation (Donaldson et al., 2012 and Foulds et al., 2012).
The general procedure reported here is that 2002 tube-format method, now with a 9 h total incubation, and optimized especially for low-end sensitivity. The specific details here are the state of the art now practiced by the Louis Picker Lab, at the Oregon Health and Science University, affiliated with the Oregon National Primate Research Center. These methods have been used in several of our recent publications (Fukazawa et al., 2012; Hansen et al., 2011; Hansen et al., 2009). It is important to note that in our hands, plate-format ICS is not as reliable or sensitive for weak responses as is this tube-based method (unpublished observations). Until that problem is understood and solved, the tube-based method remains the most-sensitive format for CFC.
Materials and Reagents
Data required: Flow cytometry data files, from CFC assay samples as described in “From Assay Set-up to Data Acquisition” (Sylwester et al., 2014).
Equipment
Incubator for tissue culture (humidified, stable at 37 °C, 5% CO2 atmosphere)
Flow cytometry analyser
Software
Software-equipped workstation, for analysis of cytometry files
e.g. FlowJo (Tree Star)
e.g. SPICE (NIAID)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sylwester, A. W., Hansen, S. G. and Picker, L. J. (2014). Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): Analysis of Flow Data. Bio-protocol 4(8): e1109. DOI: 10.21769/BioProtoc.1109.
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Category
Immunology > Immune cell function > Antigen-specific response
Immunology > Immune cell staining > Flow cytometry
Cell Biology > Cell-based analysis > Flow cytometry
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1,110 | https://bio-protocol.org/exchange/protocoldetail?id=1110&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): from Assay Set-up to Data Acquisition
Andrew W. Sylwester
SH Scott G. Hansen
LP Louis J. Picker
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1110 Views: 15974
Reviewed by: Jia Li
Original Research Article:
The authors used this protocol in Nov 2012
Download PDF
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Original research article
The authors used this protocol in:
Nov 2012
Abstract
What was initially termed ‘CFC’ (Cytokine Flow Cytometry’) is now more commonly known as ‘ICS’ (Intra Cellular Staining), or less commonly as ‘ICCS’ (Intra Cellular Cytokine Staining). The key innovations were use of an effective permeant (allowing intracellular staining), and a reagent to disrupt secretion (trapping cytokines, thereby enabling accumulation of detectable intracellular signal). Because not all researchers who use the technique are interested in cytokines, the ‘ICS’ term has gained favor, though ‘CFC’ will be used here.
CFC is a test of cell function, exposing lymphocytes to antigen in culture, then measuring any cytokine responses elicited. Test cultures are processed so as to stain cells with monoclonal antibodies tagged with fluorescent markers, and to chemically fix the cells and decontaminate the samples, using paraformaldehyde.
CFC provides the powers of flow cytometry, which includes bulk sampling and multi-parametric cross-correlation, to the analysis of antigen-specific memory responses. A researcher using CFC is able to phenotypically characterize cells cultured with test antigen, and for phenotypic subsets (e.g. CD4+ or CD8+ T cells) determine the % frequency producing cytokine above background level.
In contrast to ELISPOT and Luminex methods, CFC can correlate production of multiple cytokines from particular, phenotypically-characterized cells. The CFC assay is useful for detecting that an individual has had an antigen exposure (as in population screenings), or for following the emergence and persistence of antigen memories (as in studies of vaccination, infections, or pathogenesis). In addition to quantifying the % frequency of antigen-responding cells, mean fluorescence intensity can be used to assess how much of a cytokine is generated within responding cells.
With the technological advance of flow cytometry, a current user of CFC often has access to 11 fluorescent channels (or even 18), making it possible to either highly-characterize the phenotypes of antigen-responding cells, or else simultaneously quantify the responses according to many cytokines or activation markers. Powerful software like FlowJo (TreeStar) and SPICE (NIAID) can be used to analyse the data, and to do sophisticated multivariate analysis of cytokine responses.
The method described here is customized for cells from Rhesus macaque monkeys, and the extensive annotating notes represent a decade of accumulated technical experience. The same scheme is readily applicable to other mammalian cells (e.g. human or mouse), though the exact antibody clones will differ according to host system. The basic method described here incubates 1 x 106 Lymphocytes in 1 ml tube culture with antigen and co-stimulatory antibodies in the presence of Brefeldin A, prior to staining and fixation.
Keywords: ICS CFC Cytometry ICCS Antigen
[Historical Background] The first report of fixing and permeabilizing lymphocytes, then staining them with antibodies against IFN gamma, was made by Andersson et al. in 1989. In 1991, Sander et al. demonstrated improved methods, using paraformaldehyde to fix cells, saponin (an amphipathic glycoside) to permeabilize them, and fluorescently-labeled antibodies to stain intracellular cytokines for microscope examination. In 1993, Jung et al. extended this method for use with flow cytometry, included monensin (a polyether antibiotic ionophore which blocks intracellular protein transport) to inhibit secretion, so as to increase the intracellular signal of the cytokine molecules that would otherwise be released soon after synthesis. In 1995, Prussin and Metcalfe used directly-conjugated antibodies, and reported good results with 6 h incubations. Also in 1995, Picker et al. considerably enhanced the sensitivity and reproducibility of cytokine detection by using Brefeldin A (‘BfA’, a fungal lactone antibiotic) to block the cytokine-secretion apparatus, and by using a different permeant (Tween-20). This improved method was applied by Picker et al. in a 1997 report of the antigen-specific homeostatic mechanism in human HIV+ patients. In 2001, Schuerwegh et al. confirmed that BfA provides better cytokine signal in the assay than does monensin, though monensin is still used widely by others in this method.
Regarding non-human primate studies, two reports in 1989, one by Gardner and another by McClure, showed that Rhesus macaques were a useful model for studying HIV disease and AIDS. In 2002, Picker et al. reported the application of a specially-modified CFC assay to Rhesus macaques. In 2012, a consortium-appointed group aiming to establish standards for collaborating groups using CFC in Rhesus vaccine studies published their recommendations for a 96-well plate method with a 6 h total incubation (Donaldson et al., 2012; Foulds et al., 2012).
The general procedure reported here is that 2002 tube-format (see Note 1) method, now with a 9 h total incubation, and optimized especially for low-end sensitivity. The specific details here are the state of the art now practiced by the Picker Lab, at the Oregon Health and Science University, affiliated with the Oregon National Primate Research Center. These methods have been used in several of our recent publications (Hansen et al., 2013a; Hansen et al., 2013b, Fukazawa et al., 2012; Hansen et al., 2011; Hansen et al., 2009). It is important to note that in our hands, plate-format CFC is not as sensitive and reproducible for weak responses as is this tube-based method described here (unpublished observations). Until that difference is understood and solved, the tube-based method remains the most-sensitive format for CFC.
Materials and Reagents
Lymphocyte suspension from Rhesus macaques blood (Note 2) or bronchoaveolar lavage (BAL), or harvest from solid biopsy or necropsy tissue, cell density determined by a method accurate for the sample (Notes 3 and 4)
Need ~1 x 106 viable lymphocytes per test
Freshly-obtained (Note 5)
OR thawed cryopreserved sample (Note 6)
Antigen
Negative control(s) (Note 9)
Positive control:
Superantigen Staphylococcus Enterotoxin B SEB (Note 10) (Toxin Technology, catalog number: BT202 ) (lyophilized powder, 100 μg; stock: 100 μg/ml in water; usage: 2 μl/test)
Other positive control (experiment-specific)
Peptide mixes (1-100 different peptides, at ≥ 2 μg/peptide/1 ml-test) 15 amino acid peptides (15 mers) overlapping by 11 amino acids
Antibody
Unconjugated antibody for costimulation during culture incubation (Note 11)
Anti-CD28, pure unconjugated, clone CD29.2 (Note 12)
Anti-CD49d, pure unconjugated, clone 9F10
Stocks diluted to 0.5 mg/ml; use 1 μl per 1 x 106 Ly
Essential fluorophore-conjugated monoclonal antibodies
The fluorophores you use are dependent upon the flow cytometer available to you. Many companies sell the appropriate fluorophore-conjugated antibodies, including BD, Beckman Coulter, Life Technologies, InvitrogenTM, eBiosciences, and many others.
Anti-CD3e, clones reactive with Rhesus (SP34-2, FN18)
Anti-CD4 (L200, MT477))
Anti-CD8a (SK1, RPA-T8)
Anti-CD69 (FN50, CH/4, TP1.55.3) (Note 13)
Anti-IFNg (B27)
Anti-TNFa (MAB11)
Optional fluorophore-conjugated monoclonal antibodies
Anti-CD45 (DO58-1283) (Note 14)
Anti-IL2 (MQ1-17H12)
Anti-MIP1b (D21-1351)
Ant-CD107 (alpha: H4A3, beta: H4B4) (Note 15)
Anti-CD95 (DX2)
Anti- CD45RA (L48, 5H9, MEM-56, others) (Note 41)
Anti-CCR7 (CD197) (Note 42)
Anti-Ki67 (B56) (Note 38)
Brefeldin A (Sigma-Aldrich, catalog number: B-7651 )
Vendors: (Sigma-Aldrich, catalog number: B-7651; BioLegend, catalog number: 91850 )
Working stock: 10 mg/ml, in DMSO (1.0 μl/test) (Note 16)
Benzonase (Merck KgaA, Novagen) (use at 50 U/ml)
1x RPMI-1640 (w/o L-glutamine, 0.1 μm filtered) (e.g. HyCone, catalog number: SH30096.02 )
Fetal Bovine Serum (FBS, aka: 'FCS') (e.g. HyClone, catalog number: SH30070.03 ) (defined, heat-inactivated, 40 nm-filtered)
Penicillin+Streptomycin (P/S) Solution (e.g. Sigma-Aldrich, catalog number: P-0781 )
L-glutamine (200 mM) (e.g. Sigma-Aldrich, catalog number: G-7513 (100 ml)
Sodium pyruvate (SP) (e.g. Sigma-Aldrich, catalog number: S-8636 ) (100 ml)
Beta-Mercaptoethanol (bME) (e.g. Sigma-Aldrich, catalog number: M-7522 ) (100 ml) (Note 40)
Sterile-filtration apparatus (e.g. Corning, catalog number: 430769 ) (500 ml capacity 0.22 μm cellulose-acetate filter)
Dulbecco's Phosphate Buffered Saline (DPBS) (e.g. Thermo Fisher Scientific, Corning, catalog number: 55-031-PB )
Bovine serum albumin (BSA) (e.g. Thermo Fisher Scientific, catalog number: BP1605100 )
Sodium azide (preservative; NaN3) (e.g. Thermo Fisher Scientific, catalog number: BP922-500 )
BD FACS Lysing Solution (10x concentrate) (BD, catalog number: 349202 )
Tween-20 (polyoxyethylenesorbitan monolaurate) (e.g. Sigma-Aldrich, catalog number: P-7949 )
Aqua LIVE/DEAD kit (Life Technologies, InvitrogenTM, www.lifetechnologies.com, search 'LIVE/DEAD' for an evolving array of stains and kits) (Note 43)
Concentrated dye stock (see Recipes)
Staining Solution (made fresh) (see Recipes)
Tissue culture medium ('R10') (see Recipes)
'PAB' wash buffer (see Recipes)
'Lyse' fixation and RBC-lysing solution (see Recipes) (Note 17)
'Perm' fixation and cell-permeabilizing solution (see Recipes) (Note 18)
Equipment
Tubes (Notes 1 and 7) polypropylene (PP) (round-bottom, 5 ml/12 x 75 mm, sterile) (e.g. Falcon®, catalog number: 35-2054 )
Computer-generated printed labels for tubes (optional; must stick well to polypropylene)
Tube-holding racks (optional) (e.g. Thermo Fisher Scientific, No-Wire Grip Rack 10-13 MM 90 place) (Note 8)
Foam cosmetic wedges (or functional equivalent) (The ones we use are 2" long x ¾" high when lying on the long side.)
Laminar flow biosafety cabinet (for sterility, even if not working with pathogens)
Trapped vacuum aspirator
Appropriate fluid measuring dispensers, with appropriate disposables
Electric pump pipettors for measurements between 1-50 ml
Manual hand micropipettors for measurements between 0.5-1,000 μl
Repeater-dispensers (e.g. from Eppendorf)
Hand repeaters, for measurements between 0.5-2 ml
Stationary pump dispensers, for measurements between 1-5 ml
Centrifuge with swing-buckets (capable of 800 x g) (e.g. Sorvall Legend T/RT)
Vortexer
Lab timer
Incubator for tissue culture (humidified, stable at 37 °C, 5% CO2 atmosphere)
Option: Standard water-jacketed T/C incubator (e.g. Thermo Fisher Scientific, FormaTM, Series II, Model: 3110 )
Option: UniBator (Tritech Research) DigiTherm CO2 incubator with rapid cooling and bi-directional interface (Note 19)
Refrigerator at 4 °C
Flow cytometry analyser, 6-fluorescence detectors or more (e.g. BD, model: LSR-II )
A method of counting PBMC (e.g., Coulter counter, Guava, or hemocytometer)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sylwester, A. W., Hansen, S. G. and Picker, L. J. (2014). Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): from Assay Set-up to Data Acquisition. Bio-protocol 4(8): e1110. DOI: 10.21769/BioProtoc.1110.
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Category
Immunology > Immune cell function > Antigen-specific response
Immunology > Immune cell staining > Flow cytometry
Cell Biology > Cell-based analysis > Flow cytometry
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1,111 | https://bio-protocol.org/exchange/protocoldetail?id=1111&type=0 | # Bio-Protocol Content
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Peer-reviewed
The BhbA Enzyme Assay
KC Kai Chen
Jiandong Jiang
Published: Vol 4, Iss 8, Apr 20, 2014
DOI: 10.21769/BioProtoc.1111 Views: 8364
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
Reductive dehalogenation has been found primarily in anaerobic communities and is originally thought to rarely occur in aerobes. A reductive dehalogenase (BhbA) was characterized from an aerobic strain of Comamonas sp. 7D-2, which was isolated from a bromoxynil octanoate-contaminated soil sample collected in Jiangsu, China. BhbA catalyzes the reductive dehalogenation of bromoxynil and its derivative 3,5-dibromo-4-hydroxybenzoate under aerobic conditions. BhbA is membrane-associated and found to have the key features of anaerobic respiratory reductive dehalogenases. This protocol describes the method for enzyme analysis of the aerobic reductive dehalogenase (BhbA) in the membrane fraction.
Materials and Reagents
Comamonas sp. 7D-2
LB medium
Substrate, 3,5-dibromo-4-hydroxybenzoate (DBHB) or 3-bromo-4-hydroxybenzoate (BHB) (Sigma-Aldrich)
Electron donor, NADPH or NADH (Sangon Biotech, catalog numbers: Y4433000-100 mg and NB0642-1 g , respectively)
Reaction inhibitor, sodium dithionite (Na2S2O4) (Sigma-Aldrich, catalog number: 7775-14-6 )
Protein Quantification Kit (Sangon Biotech, catalog number: BE530-100 ml )
Phosphate buffered saline (PBS) (Sambrook and Russell, 2001) (see Recipes)
Mobile phase of HPLC (see Recipes)
Equipment
7 ml centrifuge tube
Membrane filtration (pore size, 0.22 μm)
Centrifuge
HPLC (600 controller, Rheodyne 7725i manual injector and 2487 Dual λ Absorbance Detector) (Waters)
Ultrasonic instrument
Fast protein liquid chromatography
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, K. and Jiang, J. (2014). The BhbA Enzyme Assay. Bio-protocol 4(8): e1111. DOI: 10.21769/BioProtoc.1111.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Activity
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1,112 | https://bio-protocol.org/exchange/protocoldetail?id=1112&type=0 | # Bio-Protocol Content
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Amino Acid Racemase Enzyme Assays
Atanas D. Radkov
Luke A. Moe
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1112 Views: 9140
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The authors used this protocol in Nov 2013
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Abstract
Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide a detailed protocol using high performance liquid chromatography (HPLC) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, also Marfey’s reagent) for the characterization of novel amino acid racemases. The protocol described here was designed to obtain accurate kinetic parameters (kcat, KM values). Enzyme concentrations and reaction times were optimized so as to minimize the reverse reaction, which can confound results when measuring racemase reactions.
Materials and Reagents
Escherichia coli (E. coli) Rosetta 2 (DE3) cell line
pET overexpression system
His-tag and Ni2+-NTA affinity chromatography (HIS-Select Nickel Affinity Gel) (Sigma-Aldrich, catalog number: P6611 )
AA substrates
Note: Each enantiomer of the 19 chiral proteinogenic AAs, and the four epimers of hydroxyproline, are prepared in 50 mM HEPES buffer (pH 7.4) with any co-factors (PLP at 20 μM was used in our case.).
0.5% solution (w/v, in acetone) of Marfey’s reagent (Sigma-Aldrich, catalog number: 71478 )
1 M NaHCO3
2 M HCl
2 M NaOH
HPLC buffer (0.05 M TEAP buffer, pH 3.0) (see Recipes)
Equipment
Bottle top filter (polystyrene) (Corning, catalog number: 430513 ) (filter used in TEAP buffer preparation)
Syringe filter (PTFE) (Tisch Scientific, catalog number: SF14466 ) (filter used in HPLC sample preparation)
Heat block (set at 37 °C for enzyme reactions)
Heat block (set at 40 °C for derivatization of reaction products)
2 ml microfuge tubes (for the reaction, derivatization, and dilution of derivatized products)
Amber HPLC vials with caps, syringes (1 ml), needles, and filtersfilters (0.22 μm polytetrafluoroethylene, PTFE)
Waters Nova-Pak (C18 column) (3.9 mm by 150 mm)
Software
Microsoft Excel
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Radkov, A. D. and Moe, L. A. (2014). Amino Acid Racemase Enzyme Assays. Bio-protocol 4(9): e1112. DOI: 10.21769/BioProtoc.1112.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Activity
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1,113 | https://bio-protocol.org/exchange/protocoldetail?id=1113&type=0 | # Bio-Protocol Content
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Extraction and Quantification of Poly P, Poly P Analysis by Urea-PAGE
MG Maria R. Gomez Garcia
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1113 Views: 9222
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
Inorganic polyphosphate (poly P) molecules, linear chains containing hundreds of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds are abundant in every cell in nature. These molecules are widely distributed among bacteria, including key pathogens, and eukaryotes, poly P is present in organelles, including nuclei, mitochondria, and vesicles.
Remarkable properties of this molecule as a polyanion have been discovered and have made it suited for a crucial role in the emergence of cells on earth. Poly P is essential for bacterial responses to stresses and starvation, motility, quorum sensing, biofilm formation, and virulence and essential for survival. Polymers of different lengths are present in different locations and have different roles in the cell.
Keywords: Poly P Poly P Urea-PAGE Analysis Poly P Quantification
Materials and Reagents
[γ-32P] ATP, [32P] (Amersham Biosciences)
Poly P (types 15 and 75) (apyrase, and common chemicals) (Sigma-Aldrich)
Poly P35, Poly P50, Poly P300, and Poly P750
Polyphosphatase (PPX) isolated from yeast
Polyphosphate kinase (PPK) isolated from Escherichia coli (E. coli) (EcPPK)
Tris-HCl (pH 8.0)
Ammonium sulfate
Toluidine blue
25% methanol
5% glycerol
5x sample buffer (see Recipes)
THK buffer (see Recipes)
Elution buffer (see Recipes)
Aacrylamide solution (see Recipes)
Equipment
DE81 disk
Monolight 2010 (Analytical Luminescence Laboratory)
Topcount (Packard Instruments)
Microfuge (14,000 rpm or 16,000 x g)
Phosphorimager (Molecular Dynamics) (conventional)
Kodak X-Omat AR film
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Garcia, M. R. G. (2014). Extraction and Quantification of Poly P, Poly P Analysis by Urea-PAGE. Bio-protocol 4(9): e1113. DOI: 10.21769/BioProtoc.1113.
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Category
Microbiology > Microbial biochemistry > Other compound
Biochemistry > Other compound > Ion
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1,114 | https://bio-protocol.org/exchange/protocoldetail?id=1114&type=0 | # Bio-Protocol Content
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Peer-reviewed
Detection of Tumor Cell Surface-reactive Antibodies
BA Brian M. Andersen
Michelle R. Goulart
MO Michael R. Olin
G. Elizabeth Pluhar
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1114 Views: 7996
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in May 2013
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May 2013
Abstract
Vaccine-based immunotherapy is being used to treat dogs with primary brain tumors. The vaccines are composed of a lysate of autologous tumor cells, which stimulate an immune response producing tumor specific antibodies that are capable of inducing antibody-dependent cell-mediated cytotoxicity to allogeneic, as well as autologous, tumor cells. This protocol will describe the tumor cell serum antibody-binding assay to measure the tumor-reactive IgG antibody response. Key features of this assay are that it is performed with sera collected from the canine patient prior to and following vaccination as the source of antibodies and canine brain tumor cells used as the target cells.
Materials and Reagents
Tumor cells
Note: Tumor sample is harvested from pet dogs during brain surgery.
BD MatrigelTM (BD Bioscience, catalog number: 354230 )
DMEM/F12 Medium with L-glutamine (Life Technologies, Gibco®, catalog number: 11320-033 )
50x B-27 supplement without vitamin A (Life Technologies, Gibco®, catalog number: 12587-010 )
100x N2 supplement (Life Technologies, Gibco®, catalog number: 17502-048 )
Recombinant human Epidermal Growth Factor (Pepro Tech, catalog number: AF-100-15 )
Recombinant human fibroblast growth factor-basic (Pepro Tech, catalog number: 100-18B )
50x Penicillin-streptomycin solution (Mediatech, Cellgro®, catalog number: 30-001-CI )
Dispase (BD Bioscience, catalog number: 354235 )
1x Phosphate-Buffered Saline (Mediatech, Cellgro®, catalog number: 21-040-CV )
MACS BSA stock solution (10% BSA) (Miltenyi Biotec, catalog number: 130-091-376 )
1x TrypLETM Express (Life Technologies, Gibco®, catalog number: 12605-010 )
0.4% Trypan Blue Solution (Life Technologies, Gibco®, catalog number: 15250-061 )
Goat anti-canine IgG (H&L) f(ab’)2-fluorescein isothiocyanate (American Qualex, catalog number: F145FN )
Neural Stem Cell (NSC) medium (see Recipes)
Matrigel Coating solution (see Recipes)
rh-EGF stock solution (see Recipes)
rh-FGF basic stock solution (see Recipes)
Equipment
10 mm culture plates (Sigma-Aldrich, Corning® Costar®, catalog number: CLS430165 )
Scalpel blade
100 μm filter (Corning Incorporated, catalog number: 352360 )
BD FACS tubes (12 x75 mm) (BD Biosciences, Falcon®, catalog number: 352235 )
15 ml tubes (BD Biosciences, Falcon®, catalog number: 352096 )
Culture dish (100 x 20 mm) (Sarstedt AG, catalog number: 83.1802 )
1.5 ml Eppendorf microcentrifuge tubes (Sigma-Aldrich, catalog number: T9661-500EA )
Neubauer hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
Incubator (5% CO2, 5% O2, 37 °C) (BioSpherix)
Shaker table (Eppendorf, New Brunswick Excella® E25 incubator shaker)
Centrifuge (Thermo Fisher Scientific, model: ST40R )
Microcentrifuge (Thermo Fisher Scientific, model: MicroCL 17 )
BD FACSCanto three-laser flow cytometer (BD)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Andersen, B. M., Goulart, M. R., Olin, M. R. and Pluhar, G. E. (2014). Detection of Tumor Cell Surface-reactive Antibodies. Bio-protocol 4(9): e1114. DOI: 10.21769/BioProtoc.1114.
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Category
Cancer Biology > Tumor immunology > Immunological assays
Cancer Biology > General technique > Biochemical assays
Immunology > Antibody analysis > Antibody function
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1,115 | https://bio-protocol.org/exchange/protocoldetail?id=1115&type=0 | # Bio-Protocol Content
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Minimal Bactericidal Concentration for Biofilms (MBC-B)
NB Nicole Billings
KR Katharina Ribbeck
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1115 Views: 12633
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Aug 2013
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Aug 2013
Abstract
A biofilm is a multicellular consortium of surface associated microbes surrounded by a hydrated, extracellular polymer matrix. The biofilm matrix plays a critical role in preventing desiccation, acquiring nutrients, and provides community protection from environmental assaults. Importantly, biofilms are significantly more resistant to antimicrobials relative to their free-swimming counterparts. The level of antimicrobial tolerance is influenced by a number of factors, including genetic/adaptive resistance mechanisms, stage of biofilm development, and pharmacokinetics of the antibiotic. Here, we describe an in vitro microtiter-based assay to quantify the minimal bactericidal concentration for biofilms (MBC-B) for short exposure times (2 h). This exposure period is significantly shorter than standard over-night and 24-hour treatments described in traditional protocols. This assay was developed to approximate the time an antibiotic is available during a one-time treatment before it is metabolized, sequestered by host proteins, or digested.
Materials and Reagents
Glycerol stock of Pseudomonas aeruginosa (P. aeruginosa) (acquired from a single colony, stored at -80 °C)
Bacto-tryptone (BD Biosciences, catalog number: 211705 )
Bacto-yeast extract (BD Biosciences, catalog number: 212750 )
Bacto-agar (BD Biosciences, catalog number: 214010 )
Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
10x PBS stock (Life Technologies, Gibco®, catalog number: 70011-044 )
Colistin sodium sulfate (Sigma-Aldrich, catalog number: C4461 )
Polymyxin sulfate salt (Sigma-Aldrich, catalog number: P4932 )
Tobramycin (Sigma-Aldrich, catalog number: T4014 )
Ciprofloxacin (Sigma-Aldrich, catalog number: 17850 )
LB medium for agar plates (see Recipes)
1% TB liquid medium (see Recipes)
Equipment
Glass beads (425-600 µm, sterile) (Sigma-Aldrich, catalog number: G8772 )
Aluminum film (sterile) (Sigma-Aldrich, catalog number: Z722642 )
15 ml polystyrene culture tubes (sterile) (VWR International, catalog number: 89497-782 )
Falcon 96 well polystyrene plates (flat-bottom, sterile with lid, non-treated) (Corning Incorporated, catalog number: 351172 )
Spectrophotometer to measure absorbance of cell culture (OD600)
37 °C shaking and static incubators
Titer plate shaker (Thermo Fisher Scientific, catalog number: 4625-Q )
Multichannel pipette (volume ranges 10 μl-200 μl)
Sterile plastic loops (1 μl) (Santa Cruz, catalog number: SC-200266 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial biofilm > Killing assay
Biochemistry > Other compound > Antimicrobial
Microbiology > Microbial cell biology > Cell viability
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1,116 | https://bio-protocol.org/exchange/protocoldetail?id=1116&type=0 | # Bio-Protocol Content
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Peer-reviewed
Microfluidic-based Time-kill Kinetic Assay
NB Nicole Billings
RR Roberto Rusconi
RS Roman Stocker
KR Katharina Ribbeck
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1116 Views: 11976
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Aug 2013
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Aug 2013
Abstract
In many environments, bacteria favor a sessile, surface-attached community lifestyle. These communities, termed biofilms, are ubiquitous among many species of bacteria. In some cases, biofilms form under flow conditions. Flow chambers, and in particular microfluidic channels, can be used to observe biofilm development and physiological effects while varying nutrient conditions, flow velocities, or introducing antimicrobials to the biofilm in real time. Here, we describe a microfluidic-based kill-kinetics assay for the observation of antimicrobial effects on biofilms under flowing conditions.
Materials and Reagents
Glycerol stock of Pseudomonas aeruginosa (P. aeruginosa) (acquired from a single colony, stored at -80 °C)
Bacto-tryptone (BD Biosciences, catalog number: 211705 )
Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
Colistin sodium sulfate (Sigma-Aldrich, catalog number: C4461 )
Polydimethylsiloxane (PDMS) (Sylgard 184 Silicone Elastomer Kit) (Dow Corning Corporation)
Petri dishes (150 mm x 15 mm) (VWR International, catalog number: 25384-326 )
100% Ethanol
Sterile dH2O
LIVE/DEAD BacLight Bacterial Viability Kit with Syto9/Propidium Iodide (Life Technologies, catalog number: L-7012 )
1% TB liquid medium (see Recipes)
Equipment
Sterile 14 ml polystyrene culture tubes (VWR International, catalog number: 60818-725 )
Sterile 1 μl plastic loops (Santa Cruz, catalog number: SC-200266 )
Spectrophotometer for absorbance readings
37 °C shaking incubator
Microfluidic channel molds (Figure 1)
Figure 1. Microfluidic channels. Each channel is 100 μm deep and 500 μm wide. The 10 channels were fabricated by depositing SU-8 2150 on silicon wafers using standard soft lithography techniques (Xia and Whitesides, 1998). This device allows for 10 parallel experiments.
SU-8 2150 (MicroChem Corp.)
Glass Slides (75 x 50 mm) (Ted Pella, catalog number: 26005 ) or cover glass (75 x 50 mm) (Ted Pella, catalog number: 260462 )
Lens paper
Desiccator with vacuum line
Exacto-knife or scalpel
5 ml syringes (BD, catalog number: 309647 )
Polyethylene tubing (ID 0.58 mm, OD 0.965) (BD, IntramedicTM, Clay Adams®, catalog number: 427411 )
Harris Unicore hole punch (1.20 mm) (Ted Pella, catalog number: 15074 )
Hand-held corona generator (Laboratory Corona Treater, Electro-Technic Products, Model: BD-20AC )
Syringe pump with 10-syringe holder adaptor (Harvard Apparatus)
Fluorescence microscope with automated focus/stage system (e.g., Nikon TE2000-E equipped with an Andor iXon-885 and a 40x long working distance objective)
Software
ImageJ
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial biofilm > Killing assay
Microbiology > Microbial cell biology > Cell imaging
Cell Biology > Cell imaging > Microfluidics
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1,117 | https://bio-protocol.org/exchange/protocoldetail?id=1117&type=0 | # Bio-Protocol Content
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Enterovirus 71 Virus Propagation and Purification
Kristin L. Shingler
Lindsey J. Organtini
Susan Hafenstein
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1117 Views: 13430
Original Research Article:
The authors used this protocol in Jul 2013
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Jul 2013
Abstract
Since its discovery in 1969, enterovirus 71 (EV71) has emerged as a serious worldwide health threat. This member of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. This is the propagation and purification procedure to produce infectious virion.
Keywords: Virus purification Gradient Picornavirus Procapsid
Materials and Reagents
Hela cells
EV71 inoculum (ATCC, www.atcc.org)
DMEM (Sigma-Aldrich, catalog number: SH3002201 )
Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: SH3039603 )
Polyethylene glycol (PEG) 8000 (Thermo Fisher Scientific, catalog number: P156-3 )
Tris-HCl
Magnesium chloride (MgCl2)
0.5 M NaCl
0.05 mg/ml DNase (Thermo Fisher Scientific, catalog number: NC9709009 )
0.1 M EDTA (pH=8.0)
Ammonium hydroxide (Sigma-Aldrich, catalog number: A5132-5 Kg )
Potassium tartrate (Sigma-Aldrich, catalog number: 25516-500 g )
100 kD cutoff spin column with 4 ml capacity (Millipore, catalog number: UFC810096 )
30% sucrose-cushion in purification buffer
Purification buffer (see Recipes)
Equipment
Cell Stacks (1 10-stack and 1 2-stack) (Thermo Fisher Scientific, catalog numbers: 12-567-303 and 12-567-301 )
Centrifuge
SLA1500 rotor
50.2ti rotor
26.3 ml red-capped Beckman tubes (Beckman Coulter, catalog number: 355618 )
SW41 rotor
Beckman Ultra-Clear centrifuge tubes (14 x 89 mm) (Beckman Coulter, catalog number: 344059 )
Chemistry ring stand with clamp
Small light source
Microcentrifuge
Procedure
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Category
Microbiology > Microbial cell biology > Cell isolation and culture
Microbiology > Microbe-host interactions > Virus
Cell Biology > Cell isolation and culture > Cell isolation
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1,118 | https://bio-protocol.org/exchange/protocoldetail?id=1118&type=0 | # Bio-Protocol Content
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Peer-reviewed
ELISA for Alpha-hemolysin (Hla) in Methicilin-resistant Staphylococcus aureus (MRSA)
Varandt Y. Khodaverdian
Menachem Shoham
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1118 Views: 10261
Original Research Article:
The authors used this protocol in Aug 2013
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Aug 2013
Abstract
Anti-virulence agents against MRSA inhibit the production of disease-causing virulence factors, such as alpha-hemolysin, but are neither bacteriostatic nor bactericidal. Here we discuss a rapid method to screen for MRSA anti-virulence agents by measuring alpha-hemolysin production through ELISA. This protocol can be used with other alpha-hemolysin producing bacteria or for other excreted toxins to which antibodies exist.
Materials and Reagents
Staphylococcus aureus subsp. aureus USA300
Dry ice
Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128-1 )
Polyclonal anti-Hla antibody (Abcam, catalog number: ab15948 )
Anti-alpha hemolysin (Hla) antibody conjugated to horseradish peroxidase (Abcam, catalog number: ab15949 )
3,3’,5,5’ tetramethylbenzidine (TMB) (Sigma-Aldrich, catalog number: T4444 )
Stop reagent (Sigma-Aldrich, catalog number: S5689 )
Luria broth (LB) agar plates
Phosphate buffered saline (PBS) (pH 7.2) (Sigma-Aldrich, catalog number: P5119 )
10 mg/ml Bovine serum albumin (BSA) in PBS (Sigma-Aldrich, catalog number: P3688 ) (see Recipes)
0.05% Tween 20 in PBS (Sigma-Aldrich, catalog number: P3563 ) (see Recipes)
Trypticase Soy Broth (TSB) (see Recipes)
Equipment
-80 °C freezer
14 ml polypropylene culture tube (BD, catalog number: 352059 )
Cotton ball or spill resistant caps
Microtiter 96 well EIA/RIA plates (Corning, CostarTM, catalog number: 9017 )
Microplate reader (Molecular Devices, model: SPECTRA Max M2), or any other model capable of measuring a 96 well microtiter plate at OD 650 nm
Note: If a different model of microplate reader is used, ensure appropriate model of microtiter 96-well plate is used as the model above may not work.
37 °C shaker
Nitrile gloves
Multichannel pipetman (Eppendorf)
0.22 µm syringe filter (PVDF) (Thermo Fisher Scientific, FisherbrandTM, catalog number: 09-720-3 )
3 ml luer lock syringe (BD, catalog number: 309657 )
1.5 inch needle (BD, catalog number: 305187 )
2 ml cryotubes
Plastic wrap
Disposable inoculation loops (10 µl, PS, sterile, yellow) (LPS, catalog number: M122002 )
Software
Microsoft Excel or other data processing program
Procedure
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How to cite：Khodaverdian, V. Y. and Shoham, M. (2014). ELISA for Alpha-hemolysin (Hla) in Methicilin-resistant Staphylococcus aureus (MRSA). Bio-protocol 4(9): e1118. DOI: 10.21769/BioProtoc.1118.
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Category
Microbiology > Microbial cell biology > Cell viability
Microbiology > Antimicrobial assay > Antibacterial assay
Biochemistry > Protein > Immunodetection
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1,119 | https://bio-protocol.org/exchange/protocoldetail?id=1119&type=0 | # Bio-Protocol Content
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Peer-reviewed
Immunostaining Protocol: P-Smad2 (Xenograft and Mice)
AC Alexandre Calon
EE Elisa Espinet
SP Sergio Palomo-Ponce
DT Daniele V. F. Tauriello
MI Mar Iglesias
MC María Virtudes Céspedes
MS Marta Sevillano
CN Cristina Nadal
PJ Peter Jung
XZ Xiang H.-F. Zhang
DB Daniel Byrom
AR Antoni Riera
DR David Rossell
RM Ramón Mangues
JM Joan Massague
ES Elena Sancho
EB Eduard Batlle
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1119 Views: 8018
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Nov 2012
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Nov 2012
Abstract
Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.
Materials and Reagents
Paraffin sections (subcutaneous tumors samples or liver metastasis from nude mice respectively injected subcutaneously or intrasplenic with CRC cells)
XILOL
Note: Xylol also referred to as xylene or dimethylbenzene is a solvent used in histology as a clearing agent to remove paraffin from dried microscope slides prior to staining.
MilliQ H2O
Wash buffer (Dako, catalog number: K800721 )
Rabbit anti-P-Smad2 (Cell Signaling Technology, catalog number: 3108 )
BrightVision poly-HRP anti- Rabbit (Immunologic, catalog number: DPVR-110HRP )
Envision FLEX antibody diluent (Dako, catalog number: K8006 )
Peroxidase Blocking Solution (Dako, catalog number: S202386 )
ImmPACT DAB (Vector Laboratories, catalog number: SK-4105 )
DPX mounting media (Sigma-Aldrich, catalog number: 06522 )
Hematoxylin
Citrate buffer (pH 6) (see Recipes)
Equipment
Oven
Immunostaining apparatus
Autoclave
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cancer Biology > Invasion & metastasis > Animal models
Cell Biology > Cell imaging > Fluorescence
Biochemistry > Protein > Immunodetection
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1,120 | https://bio-protocol.org/exchange/protocoldetail?id=1120&type=0 | # Bio-Protocol Content
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Immunostaining Protocol: P-Stat3 (Xenograft and Mice)
AC Alexandre Calon
EE Elisa Espinet
SP Sergio Palomo-Ponce
DT Daniele V. F. Tauriello
MI Mar Iglesias
MC María Virtudes Céspedes
MS Marta Sevillano
CN Cristina Nadal
PJ Peter Jung
XZ Xiang H.-F. Zhang
DB Daniel Byrom
AR Antoni Riera
DR David Rossell
RM Ramón Mangues
JM Joan Massague
ES Elena Sancho
EB Eduard Batlle
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1120 Views: 10301
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Nov 2012
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The authors used this protocol in:
Nov 2012
Abstract
We sought to understand the mechanisms behind the potent effect of stromal TGF-beta program on the capacity of colorectal cancer (CRC) cells to initiate metastasis. We discovered that mice subcutaneous tumors and metastases generated in the context of a TGF-beta activated microenvironment displayed prominent accumulation of p-STAT3 in CRC cells compared with those derived from control cells. STAT3 signaling depended on GP130 as shown by strong reduction of epithelial p STAT3 levels upon GP130 shRNA-mediated knockdown in CRC cells.
Materials and Reagents
Paraffin sections (subcutaneous tumors samples or liver metastasis from nude mice respectively injected subcutaneously or intrasplenic with CRC cells)
XILOL
Note: Xylol also referred to as xylene or dimethylbenzene is a solvent used in histology as a clearing agent to remove paraffin from dried microscope slides prior to staining.
MilliQ H2O
Wash buffer (Dako, catalog number: K800721 )
Rabbit anti-P-Stat3 (Cell Signaling Technology, catalog number: 9145S )
BrightVision poly-HRP anti- Rabbit (Immunologic, catalog number: DPVR110HRP )
Envision FLEX antibody diluent (Dako, catalog number: K8006 )
Peroxidase Blocking Solution (Dako, catalog number: S202386 )
ImmPACT DAB (Vector Laboratories, catalog number: SK-4105 )
DPX mounting media (Sigma-Aldrich, catalog number: 06522 )
Hematoxylin
Tris/EDTA (pH 9.0) (see Recipes)
Equipment
Oven
Immunostaining apparatus
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Calon, A., Espinet, E., Palomo-Ponce, S., Tauriello, D. V. F., Iglesias, M., Céspedes, M. V., Sevillano, M., Nadal, C., Jung, P., Zhang, X. H., Byrom, D., Riera, A., Rossell, D., Mangues, R., Massague, J., Sancho, E. and Batlle, E. (2014). Immunostaining Protocol: P-Stat3 (Xenograft and Mice). Bio-protocol 4(9): e1120. DOI: 10.21769/BioProtoc.1120.
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Category
Cancer Biology > General technique > Biochemical assays
Cell Biology > Tissue analysis > Tissue staining
Biochemistry > Protein > Immunodetection
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1,121 | https://bio-protocol.org/exchange/protocoldetail?id=1121&type=0 | # Bio-Protocol Content
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Peer-reviewed
Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells
Mackenzie K. Herroon
Izabela Podgorski
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1121 Views: 22535
Edited by: Lin Fang
Reviewed by: Fanglian HeHui Zhu
Original Research Article:
The authors used this protocol in Mar 2013
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Mar 2013
Abstract
Bone is a primary site of metastasis from prostate and breast cancers. Bone marrow macrophages (BMMs) are mediators of inflammatory processes and are thought to promote tumor growth in the skeletal sites. In order to elucidate how their interactions with tumor cells impact aggressiveness of metastatic tumors in bone in vitro methods are required. By employing a system in which BMMs and tumor cells are grown separately, yet share the media and exchange soluble factors, contribution of each cell type in the context of BMM-tumor cell relationship in the bone marrow can be investigated. Additional advantages of this system include the ability to study: 1) phenotypic changes in BMMs and tumor cells upon co-culture; 2) cell-specific modulation of protein and gene expression; and 3) effects on proliferation and cell survival. It is noteworthy, that this transwell co-culture system is not limited to BMMs and tumor cells and can be easily modified to include other components of bone marrow microenvironment (e.g., adipocytes, stromal cells, osteoblasts).
Materials and Reagents
Mice (FvBN strain)
PC3 cells (ATCC, catalog number: CRL 7934 )
L929 cells (ATCC, catalog number: VR-1404 )
DMEM (Sigma-Aldrich, catalog number: D2902 )
MEM-alpha (Sigma-Aldrich, catalog number: M0644 )
Fetal bovine serum (Thermo Fisher Scientific, HyClone catalog number: SH3008803 )
Pencillin-Streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
0.25% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25200-056 )
1x Sterile PBS (Sigma-Aldrich, catalog number: P3813 )
70% ethanol
RNeasy Plus Mini Kit (QIAGEN, catalog number: 74134 )
QIAshredder (QIAGEN, catalog number: 79654 )
RLT buffer (part of RNeasy Plus Mini Kit, shown above), includes 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
Sucrose (Sigma-Aldrich, catalog number: S9378 )
MES (Sigma-Aldrich, catalog number: M8250 )
Complete DMEM (see Recipes)
L929 conditioned media (see Recipes)
BMM media (see Recipes)
SME lysis buffer (see Recipes)
Equipment
6-well plates (Corning, Costar®, catalog number: 3516 )
Transwell inserts (Corning, catalog number: 3412 )
Petri dishes (Thermo Fisher Scientific, catalog number: 08-757-13 )
100 mm tissue culture dishes (Corning, catalog number: 430293 )
T75 flasks (Corning, catalog number: 430725 )
Cell scrapers (SARSTEDT AG, catalog number: 83.1830 )
Sterile fine-tip transfer pipets (Thermo Fisher Scientific, catalog number: 232-1S )
10 ml syringes (Luer-Lok) (BD, catalog number: 309604 )
5, 10, & 25 ml serological pipets
Needles (26 Gauge & 20 Gauge) (BD, catalog numbers: 305111 & 305176 , respectively)
Dissecting forceps and scissors
Scalpel
1.5 ml tubes (Thermo Fisher Scientific, catalog number: 3464 )
15 ml centrifuge tubes (Thermo Fisher Scientific, Nunc®, catalog number: 339650 )
50 ml centrifuge tubes (Thermo Fisher Scientific, Nunc®, catalog number: 339652 )
5 K Millipore centrifugal concentrator (Amicon Ultra-4) (Millipore, model: UFC800596 )
Centrifuge
Incubator (5% CO2, 37 °C) (Napco Series 8000 DH CO2 Incubator)
Biosafety cabinet (The Baker Company, SterilGARD®)
Procedure
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Category
Cancer Biology > General technique > Cell biology assays
Cancer Biology > Tumor immunology > Tumor microenvironment
Cell Biology > Tissue analysis > Tissue isolation
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1,122 | https://bio-protocol.org/exchange/protocoldetail?id=1122&type=0 | # Bio-Protocol Content
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Extraction and Measurement the Activities of Cytosolic Phosphoenolpyruvate Carboxykinase (PEPCK) and Plastidic NADP-dependent Malic Enzyme (ME) on Tomato (Solanum lycopersicum)
SO Sonia Osorio
JV José G. Vallarino
MS Marek Szecowka
SU Shai Ufaz
VT Vered Tzin
RA Ruthie Angelovici
GG Gad Galili
AF Alisdair R. Fernie
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1122 Views: 10368
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Feb 2013
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Feb 2013
Abstract
A recent study demonstrated that cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and NADP-malic enzyme (NADP-ME) have an important role in malate metabolism during fruit ripening (Osorio et al., 2013). PEPCK catalyze the ATP-dependent decarboxylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and NADP-ME, the reversible conversion of malate and pyruvate. Here, we present the detailed protocols to measure PEPCK activity in carboxylation direction by following oxidation of NADH and to measure NADP-ME activity based upon the reduction of NADP+.
Materials and Reagents
PEPCK activity
Un-harvested plant tissues (how to freeze the tissue is explained in the procedure section)
Liquid N2
Ice
Bicine (Sigma-Aldrich, catalog number: B8660 )
KOH
EDTA (Sigma-Aldrich, catalog number: EDS-100G )
Poly (ethylene glycol)-4000 (Sigma-Aldrich, catalog number: 81240 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
β-nicotinamide adenine dinucleotide reduced form (NADH) (Roche Diagnostics, catalog number: 10128015001 )
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
KCl
MnCl2
Phosphoelnolpyryvate (PEP) (Bio Vectra, catalog number: 2552)
Adenosine 5′-diphosphate sodium salt (ADP) (Sigma-Aldrich, catalog number: A2754 )
KHCO3
L-Malate dehydrogenase (Roche Diagnostics, catalog number: 10127248001 )
Bradford stock solution (Bio-Rad Laboratories, catalog number, 500-0006 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
Extraction buffer 1 (see Recipes)
Extraction buffer 2 (see Recipes)
Buffer 3 (see Recipes)
PEPCK assay mix 1 (see Recipes)
NADP-ME activity
Un-harvested plant tissues (how to freeze the tissue is explained in the procedure section)
Liquid N2
Ice
Tris-Base (United State Biological, catalog number: T8600 )
MnCl2
EDTA (Sigma-Aldrich, catalog number: EDS-100G)
Glycerol (Sigma-Aldrich, catalog number: G5516 )
2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
β-nicotinamide adenine dinucleotide phosphate (NADP+) (Roche Diagnostics, catalog number: 10128058001 )
L-Malate (Sigma-Aldrich, catalog number: M1000 )
Bradford stock solution (Bio-Rad Laboratories, catalog number, 500-0006)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058)
Extraction buffer 4 (see Recipes)
NADP-ME assay mix 1 (see Recipes)
Equipment
Small mortar and pestle
2 ml and 1.5 ml microfuge tubes
Pipettes
Balance
2 ml centrifuge (Hettich Mikro 22R)
96 well polystyrene microplate (flat bottom) (Corning, catalog number: 3300 )
A computer supported microplate spectrophotometer for kinetic (time-course) measurement mode (Elisa microplate-spetrophotometer) (BioTek Instruments, model: EL808 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Isolation and purification
Plant Science > Plant biochemistry > Protein
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1,123 | https://bio-protocol.org/exchange/protocoldetail?id=1123&type=0 | # Bio-Protocol Content
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In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans
Mohlopheni J. Marakalala
Published: Vol 4, Iss 9, May 5, 2014
DOI: 10.21769/BioProtoc.1123 Views: 8268
Edited by: Fanglian He
Reviewed by: Heather Taff
Original Research Article:
The authors used this protocol in Apr 2013
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The authors used this protocol in:
Apr 2013
Abstract
Macrophage recognition of Candida albicans (C. albicans) is facilitated by pattern recognition receptors that interact with the fungal pathogen associated molecular patterns (PAMPs). Dectin-1 is the major macrophage receptor that is known to recognize fungal Beta-glucans leading to induction of various immune responses. This receptor is also known to be required for in vivo protection against C. albicans (Taylor et al., 2007). We recently showed that the Dectin-1 mediated protection in vivo is strain-dependent, and that C. albicans can adapt to modulate immune recognition by Dectin-1 (Marakalala et al., 2013). In vitro analysis, however, showed a Dectin-1-dependent and pro-inflammatory responses against all strains tested. This protocol describes in detail the in vitro analysis used in the paper. In particular, methods involved in fluorescent labeling of live C. albicans, quantification of macrophage binding of the pathogen, and pro-inflammatory responses to yeast and hyphal forms of the fungi are described.
Keywords: Candida albicans Dectin-1 Beta-glucans Macrophages Pro-inflammatory responses
Materials and Reagents
Candida albicans strains, SC5314 and ATCC18804
Mice
Note: 2x per WT or Dectin-1 KO mice on C57BL/6 background should give sufficient macrophages for a full 24-well plate experiment. Mice were age and sex matched.
Thioglycollate-elicited macrophages
Note: This method has been described in detail in Kerrigan et al. (2012).
RPMI 1640 medium (Life Technologies, catalog number: 11875-093 )
70% ethanol
Sabouraud Dextrose broth (Oxoid Limited, catalog number: CM0147 )
Rhodamine Green-X (Life Technologies, InvitrogenTM, catalog number: R-6113 )
Difco Thioglycollate Broth (BD Biosciences, catalog number: 225710 )
Triton-X 100 (Sigma-Aldrich, catalog number: T8787 )
OptEIA TNF kit (BD Biosciences, catalog number: 555268 )
Zymosan (Life Technologies, InvitrogenTM, catalog number: Z2849 )
Dulbecco’s Phosphate Buffered Saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
Fetal Bovine/Calf Serum (FCS) (Sigma-Aldrich, catalog number: F6178 )
EDTA (Sigma-Aldrich, catalog number: E9884 )
Equipment
Titer-Tek Fluoroskan II (Labsystems)
Shaker
Note: The shaker temperature was controlled at 30 °C and the speed was between 150 to 200 rpm.
Centrifuge (Eppendorf, model: 5810R with swing bucket rotor S-4-72 )
Light microscope
Haemocytometer
24-well plates (BD Biosciences)
2 ml Eppendorf tube
96-well plates (black in color for fluorescence measurement) (BD Biosciences)
Rubber back of syringe plunger
Note: The make or brands for above equipment are not important for reproducibility.
Procedure
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Copyright: © 2014 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:
Marakalala, M. J. (2014). In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans. Bio-protocol 4(9): e1123. DOI: 10.21769/BioProtoc.1123.
Marakalala, M. J., Vautier, S., Potrykus, J., Walker, L. A., Shepardson, K. M., Hopke, A., Mora-Montes, H. M., Kerrigan, A., Netea, M. G., Murray, G. I., Maccallum, D. M., Wheeler, R., Munro, C. A., Gow, N. A., Cramer, R. A., Brown, A. J. and Brown, G. D. (2013). Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1. PLoS Pathog 9(4): e1003315.
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Category
Microbiology > Microbe-host interactions > Fungus
Immunology > Host defense > Murine
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1,124 | https://bio-protocol.org/exchange/protocoldetail?id=1124&type=0 | # Bio-Protocol Content
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Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)
Toshitsugu Fujita
Hodaka Fujii
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1124 Views: 14354
Reviewed by: Sabine Le Saux
Original Research Article:
The authors used this protocol in Nov 2013
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Abstract
Elucidation of molecular mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, it is useful to isolate the target regions retaining molecular interactions. We established locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) for isolation of target genomic regions (Hoshino and Fujii, 2009; Fujita and Fujii, 2011; Fujita and Fujii, 2012; Fujita and Fujii, 2013a; Fujita and Fujii, 2013b; Fujita et al., 2013). Identification and characterization of molecules interacting with the isolated genomic regions facilitates understanding of molecular mechanisms of functions of the target genome regions.
Here, we describe enChIP, in which engineered DNA-binding molecules, such as zinc-finger proteins, transcription activator-like (TAL) proteins, and a catalytically inactive Cas9 (dCas9) plus small guide RNA (gRNA), are utilized for affinity purification of target genomic regions. The scheme of enChIP is as follows:
1. A zinc-finger protein, TAL or dCas9 plus gRNA is generated to recognize DNA sequence in a genomic region of interest.
2. The engineered DNA-binding molecule is fused with a tag(s) and the nuclear localization signal (NLS), and expressed in the cell to be analyzed.
3. The resultant cell is crosslinked, if necessary, and lysed, and DNA is fragmented.
4. The complexes including the engineered DNA-binding molecule are subjected to affinity purification such as mmunoprecipitation. The isolated complexes retain molecules interacting with the genomic region of interest.
5. Reverse crosslinking and subsequent purification of DNA, RNA, or proteins allow identification and characterization of these molecules.
In this protocol, we describe enChIP with a TAL protein to isolate a genomic region of interest and analyze the interacting proteins by mass spectrometry (Fujita et al., 2013).
Keywords: Chromatin immunoprecipitation ChIP Locus-specific ChIP EnChIP Chromatin
Materials and Reagents
Target cells
37% formaldehyde (Nacalai Tesque, catalog number: 16223-55 )
Glycine (Sigma-Aldrich, catalog number: G7126 )
NaCl (Sigma-Aldrich, catalog number: S9625 )
Agarose S (Wako Pure Chemical Industries, catalog number: 318-01195 )
1 M Tris (pH 6.8) (AppliChem GmbH, catalog number: A4987 )
1 M Tris (pH 7.5) (AppliChem GmbH, catalog number: A4263 )
1 M Tris (pH 8.0) (AppliChem GmbH, catalog number: A4577 )
0.5 M EDTA (pH 8.0) (Nacalai Tesque, catalog number: 14362-95 )
0.1 M EGTA (Nacalai Tesque, catalog number: 08947-35 )
IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
Triton X-100 (Nacalai Tesque, catalog number: 25987-85 )
10% SDS solution (Nacalai Tesque, catalog number: 30562-04 )
8 M LiCl solution (Nacalai Tesque, catalog number: 20077-84 )
2-mercaptoethanol (Nacalai Tesque, catalog number: 21438-82 )
Sucrose (Nacalai Tesque, catalog number: 30404-45 )
Bromophenol blue (Nacalai Tesque, catalog number: 05808-61 )
Tween-20 (Sigma-Aldrich, catalog number: P5927 )
Deoxycholic acid sodium salt monohydrate (Nacalai Tesque, catalog number: 10712-12 )
30% lauroylsarcosine (Nacalai Tesque, catalog number: 20135-14 )
Dynabeads-protein G (Life Technologies, catalog number: DB10004 )
Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F1804 )
Normal mouse IgG (Santa Cruz, catalog number: sc-2025 )
10x phosphate buffered saline (PBS) (pH7.4) (Nacalai Tesque, catalog number: 27575-31 )
PBS (10x dilution of 10x PBS with distilled water)
BSA fraction V (7.5%) (Life Technologies, catalog number: 15260 )
Complete, mini, EDTA-free protease inhibitor (Roche Diagnostics, catalog number: 4693159 )
10 mg/ml RNase A (Sigma-Aldrich, catalog number: R6513 )
20 mg/ml Proteinase K (Roche Diagnostics, catalog number: 3115828 )
3x FLAG peptide (Sigma-Aldrich, catalog number: F4799 )
2-propanol (Nacalai Tesque, catalog number: 29113-95 )
3 M sodium acetate buffer solution (pH 5.2) (Nacalai Tesque, catalog number: 06893-24 )
20 mg/ml glycogen (Roche Diagnostics, catalog number: 901393 )
70% ethanol
4-20% mini-PROTEAN TGX gel (10 well, 50 µl) (Bio-Rad Laboratories, catalog number: 456-1094 )
10x running buffer solution for SDS-PAGE (Tris-Glycine) (Nacalai Tesque, catalog number: 30329-74 )
Coomassie brilliant blue R-250 staining solution (Bio-Rad Laboratories, catalog number: 161-0436 )
In-Gel Tryptic Digestion Kit (Thermo Fisher Scientific, catalog number: 89871 )
1.25 M glycine solution (200 ml) (see Recipes)
10% sodium deoxycholate (10 ml) (see Recipes)
10% Tween-20 (10 ml) (see Recipes)
Cell lysis buffer (CLB) (40 ml) (see Recipes)
Nuclear lysis buffer (NLB) (40 ml) (see Recipes)
Modified lysis buffer 3 (MLB3) (10 ml) (see Recipes)
PBS-T (10 ml) (see Recipes)
PBS-T-BSA (10 ml) (see Recipes)
5% Triton X-100 (in MLB3) (5 ml) (see Recipes)
Low salt buffer (LSB) (10 ml) (see Recipes)
High salt buffer (HSB) (10 ml) (see Recipes)
LiCl buffer (20 ml) (see Recipes)
TBS-IGEPAL CA-630 (10 ml) (see Recipes)
Elution buffer (500 µl) (see Recipes)
2x sample buffer (50 ml) (see Recipes)
Equipment
Magnetic stand (magical trapper) (TOYOBO, catalog number: MGS-101 )
Centrifuge
1.5 ml centrifuge tube (Sarstedt AG, catalog number: 72.690.001 )
2 ml centrifuge tube (Eppendorf, catalog number: 0030.120.094 )
50 ml centrifuge tube (BD Biosciences, catalog number: 352070 )
Vortex mixer
Rotator
Vaccum pump connected to a side-arm flask
Sonicator (ultrasonic disruptor UD-201 ) (TOMY SEIKO, catalog number: UD-201)
Shaker
Mass spectrometry facility: a nanoLC-MS/MS system, composed of LTQ Orbitrap Velos (Thermo Fisher Scientific) coupled with nanoLC (Advance CaptiveSpray SOURCE, Michrom BioResources) and HTC-PAL autosampler (CTC Analytics)
Procedure
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Category
Molecular Biology > DNA > DNA-protein interaction
Biochemistry > Protein > Immunodetection
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1,125 | https://bio-protocol.org/exchange/protocoldetail?id=1125&type=0 | # Bio-Protocol Content
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Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells
Agnieszka Pastuła
Q Michael Quante
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1125 Views: 24927
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in May 2009
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The authors used this protocol in:
May 2009
Abstract
The intestine, together with skin and blood, belongs to the organs with the highest cell turnover, which makes it a perfect model to study cellular processes, such as proliferation and differentiation. Epithelial cell turnover in intestine is possible due to the presence of intestinal stem cells, which are located at the bottom of the crypt. Here, we recapitulate a detailed protocol for the isolation and culture procedures of primary epithelial intestinal cells in a three - dimensional (3D) in vitro system, described for the first time by Hans Clevers group (Sato et al., 2009). This specific 3D culture preserves intestinal stem cells, which give rise to differentiated progeny for example goblet cells. The culture has many applications and represents a useful model to study stem cell biology, epithelial cell regeneration, and transplantation studies. Moreover, the presented 3D culture can be used to investigate the barrier function of intestinal epithelial cells, as well as heterotypic cell interactions between epithelial cells and stromal cells.
Keywords: Intestinal epithelium Intestinal stem cells Organoid Three-dimensional cell culture Small intestine
Materials and Reagents
Mouse (4 weeks-16 weeks old)
Advanced DMEM/F12 (Life Technologies, Gibco®, catalog number: 12634-010 )
Heat Inactivated Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 10500-064 )
PBS without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14190-094 )
Penicillin/Streptomycin (10,000 Units/ml Penicillin; 10,000 µg/ml Streptomycin) (Life Technologies, Gibco®, catalog number: 15140-22 )
70% ethanol
100x GlutaMAXTM-I (Life Technologies, Gibco®, catalog number: 35050-038 )
1 M HEPES (Life Technologies, Gibco®, catalog number: 15630-080 )
50x B-27 Supplement (Life Technologies, Gibco®, catalog number: 17504-044 )
100x N-2 Supplement (Life Technologies, Gibco®, catalog number: 17502-048 )
BD Matrigel Basement Membrane Matrix (10 ml) (BD Biosciences, catalog number: 354234 )
Gentamicin Reagent Solution (50 mg/ml) (Life Technologies, catalog number: 15750-060 )
Sterile 0.1% BSA (in PBS)
Sterile 2 mM EDTA (see Recipes)
Washing solution (see Recipes)
N-Acetyl-L-cysteine (Sigma-Aldrich, catalog number: A9165-5G ) (see Recipes)
Recombinant Murine EGF (Pepro Tech, catalog number: AF-315-09 ) (see Recipes)
Recombinant Murine Noggin (Pepro Tech, catalog number: 250-38 ) (see Recipes)
Recombinant Human R-Spondin-1 (Pepro Tech, catalog number: 120-38 ) (see Recipes)
Equipment
Forceps and short, sharp-point scissors (e.g. Hardened Fine Iris Scissors, Fine Science Tools, catalog number: 14090-09 )
70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
Centrifuge 5702R (Eppendorf)
Cover glass
Microscope
24-well tissue culture plate (Sarstedt AG, catalog number: 83.1836 )
Petri dishes
Falcon tubes (50 ml, 15 ml)
Pipettes (20 ml, 10 ml, 1 ml, 100 µl)
Pipetboy
37 °C, 5% CO2 cell culture incubator
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pastuła, A. and Quante, M. (2014). Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells. Bio-protocol 4(10): e1125. DOI: 10.21769/BioProtoc.1125.
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Category
Cell Biology > Cell isolation and culture > 3D cell culture
Stem Cell > Adult stem cell > Epithelial stem cell
Cell Biology > Cell isolation and culture > Cell isolation
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1,126 | https://bio-protocol.org/exchange/protocoldetail?id=1126&type=0 | # Bio-Protocol Content
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Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV)
Stephen M. Laidlaw
Michael A. Skinner
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1126 Views: 8183
Original Research Article:
The authors used this protocol in May 2013
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May 2013
Abstract
The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.
Keywords: Knockout Fowlpox Deletion Poxvirus Transient dominant
Materials and Reagents
Primary chick embryo fibroblast cells (CEFs)
Notes:
Prepared from specific pathogen free quality embryos (10 days old)
For a protocol to prepare CEFs see http://cshprotocols.cshlp.org/content/2006/2/pdb.prot4475.full
Fowlpox virus (FWPV) (e.g. FP9 strain) (GenBank accession number: AJ581527 )
Gene specific oligonucleotides
High fidelity taq polymerase (e.g. Q5, New England Biolabs, catalog number: M0491S )
Qiaquick PCR purification kit (QIAGEN, catalog number: 28104 )
T4 DNA ligase (New England Biolabs, catalog number: M0202T )
Restriction enzymes (New England Biolabs)
199 media (Life Technologies, catalog number: 31150-022 )
DMEM (Life Technologies, catalog number: 11995-065 )
10% tryptose phosphate broth (Sigma-Aldrich, catalog number: T8159 )
Penicillin/Streptomycin (Life Technologies, catalog number: 15140122 )
Nystatin (Sigma-Aldrich, catalog number: N1638 )
NewBorn bovine serum (Life Technologies, catalog number: 16010-167 )
Poxvirus recombination vector e.g. pGNR (as described in Reference 1)
Transfection reagent e.g. Polyfect (QIAGEN, catalog number: 301105 )
Mycophenolic acid (Sigma-Aldrich, catalog number: M5255 )
Xanthine (Sigma-Aldrich, catalog number: X3627 )
Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
Low melting point agarose (Sigma-Aldrich, catalog number: A4018 )
10x MEM (Life Technologies, catalog number: 21430-020 )
L-glutamine (Life Technologies, catalog number: 25030-024 )
Sodium bicarbonate (Life Technologies, InvitrogenTM, catalog number: 25080-060 )
Wizard® SV genomic DNA purification kit (Promega Corporation, catalog number: A2361 )
Taq DNA polymerase (Life Technologies, InvitrogenTM, catalog number: 10342053 )
MXH media (see Recipes)
Overlay medium (see Recipes)
Complete 199 media (see Recipes)
Equipment
Thermal Cycler
Microcentrifuge
T25 cell culture flask
-80 °C freezer
Cell culture 6 well plates
Marker pen
37 °C 5% CO2 cell culture incubator
Sterile screw cap microfuge tubes
Pipette tips (ART® 200 G)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial genetics > Mutagenesis
Molecular Biology > DNA > Mutagenesis
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1,127 | https://bio-protocol.org/exchange/protocoldetail?id=1127&type=0 | # Bio-Protocol Content
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ELISA on Virus-Infected Cells
DG Daniel L. Glauser
PS Philip G. Stevenson
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1127 Views: 10608
Original Research Article:
The authors used this protocol in Oct 2013
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Abstract
The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion antigenicity during virus entry were first described by immunofluorescence of infected cells. Although immunofluorescence provides valuable information on the subcellular distribution of the viral glycoproteins, the quantification of immunofluorescence signals in a large number of cells is not only dependent on relatively expensive microscopy equipment, but is also relatively time-consuming. In order to quantify the antigenicity of MuHV-4 virions entering NMuMG epithelial cells in a reliable, as well as time- and cost-effective way, we have developed an ELISA with infected cells as the solid phase. In this assay, cells are grown on 96-well tissue culture plates, exposed to virions at 4 °C, followed by incubation at 37 °C allowing virion endocytosis. Cells are fixed either directly after virion binding at 4 °C or after incubation at 37 °C. After subsequent permeabilization, the cells are incubated with monoclonal antibodies specific for the viral envelope glycoproteins, followed by detection with an alkaline phosphatase-coupled secondary antibody. Upon incubation of cells with p-nitrophenyl phosphate substrate, the absorbance is measured on a conventional ELISA microplate reader. The different ways of data interpretation are discussed.
Keywords: Herpesvirus Cell entry Viral glycoproteins Murid herpesvirus 4 ELISA
Materials and Reagents
NMuMG cells (ATCC, catalog number: CRL-1636 )
BAC-derived MuHV-4 strain 68 (Adler et al., 2000)
Glutamine (PAA Laboratories GmbH, catalog number: E15-883 )
Fetal bovine serum (FBS) (Amimed, catalog number: 2-01F10-I )
100 U/ml penicillin and 100 µg/ml streptomycin (PAA Laboratories GmbH, catalog number: P11-010 )
Trypsin-EDTA (PAA Laboratories GmbH, catalog number: L11-003 )
Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
Glycine (Sigma-Aldrich, catalog number: 50046 )
Triton-X100 (Sigma-Aldrich, catalog number: X100 )
Tween 20 (Sigma-Aldrich, catalog number: P1379 )
Bovine serum albumin (BSA) (Fraction V) (Amresco, catalog number: 0332 )
MuHV-4 envelope glycoprotein-specific monoclonal antibodies (hybridoma supernatants) (Glauser et al., 2012b; Glauser et al., 2013)
Alkaline phosphatase-conjugated goat anti-mouse IgG (γ chain specific) polyclonal antibody (SouthernBiotech, catalog number: 1030-04 )
p-nitrophenyl phosphate tablets (Sigma-Aldrich, catalog number: N2770 )
Complete medium (see Recipes)
10x PBS (pH 7.4) (see Recipes)
1x PBS (pH 7.4) (see Recipes)
Trypsin-EDTA (see Recipes)
PBS (pH 7.4)
PBS containing 4% formaldehyde (see Recipes)
PBS containing 0.1 M glycine (see Recipes)
PBS containing 0.1% Triton-X100 (see Recipes)
PBS containing 0.1% Tween 20 and 2% BSA (see Recipes)
PBS containing 0.1% Tween 20 (see Recipes)
ELISA substrate (p-nitrophenyl phosphate) (see Recipes)
Equipment
150 cm2 tissue culture flasks (TPP Techno Plastic Products, catalog number: 90150 )
96-Well tissue culture plates (F-base) (TPP Techno Plastic Products, catalog number: 92096 )
Standard tissue culture equipment
Cooled tabletop microfuge
Fume hood
37 °C, 5% CO2 incubator
4 °C cold room
Sunrise microplate reader (Tecan Trading AG)
Neubauer improved hemocytometer
10, 20, 200, and 1,000 µl volume pipettes and tips
200 µl volume 12-channel pipette
5 ml Bijoux tubes (Sigma-Aldrich, catalog number: Z645346 )
Disposable sterile buffer reservoirs for multichannel pipettes
Polystyrene boxes
Software
Microsoft Excel 2010
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbe-host interactions > Virus
Biochemistry > Protein > Immunodetection
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1,128 | https://bio-protocol.org/exchange/protocoldetail?id=1128&type=0 | # Bio-Protocol Content
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Arabidopsis thaliana Embryo Sac Mitochondrial Membrane Potential Stain
María Victoria Martin
Diego Fernando Fiol
EZ Eduardo Julián Zabaleta
Gabriela Carolina Pagnussat
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1128 Views: 10688
Edited by: Ru Zhang
Reviewed by: Beery YaakovSamik Bhattacharya
Original Research Article:
The authors used this protocol in May 2013
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Abstract
The aim of this experiment is to study mitochondrial functional status in Arabidopsis embryo sacs using the membrane potential indicator JC-1. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting equilibrium of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol, a distinctive feature of the early stages of programmed cell death.
JC-1 is a lipophilic dye that can selectively enter into mitochondria and reversibly change color from green to red as the membrane potential increases. In healthy cells with high mitochondrial potential, JC-1 spontaneously forms complexes with intense red fluorescence. On the other hand, in mitochondria with low mitochondrial potential, JC-1 remains in the monomeric form, which exhibits only green fluorescence (Martin et al., 2013; Hauser et al., 2006).
This protocol could be used in isolated mitochondria, and in a variety of cell types and different tissues of plants and other organism.
Keywords: Mitochondrial membrane potential Female gametophyte Arabidopsis thaliana Plant embryo sac JC-1
Materials and Reagents
Flowers at different developmental stages from an Arabidopsis inflorescence
JC-1 dye (Life Technologies, Molecular Probes®, catalog number: T3168 )
DMSO 99.9% (Sigma-Aldrich, catalog number: D8418 )
Stock solution (10 mg/ml of JC-1 in DMSO)
Working solution (10 µg/ml of JC-1 in buffer A)
Buffer A (20 mM HEPES buffer, pH 7.2) (Sigma-Aldrich, catalog number: H3375 ) (see Recipes)
Equipment
Confocal microscope (Nikon Eclipse C1 Plus Confocal microscope, using EZ-C1 3.80 imaging software and Ti-Control)
Dissecting microscope (Nikon Corporation, model: SMZ800 )
Coverslip (18 x 18 mm)
Microscopic slide (26 x 76 mm)
1 ml insulin syringe with the 0.3 x 13 mm needle (BD)
Needle point tweezers
Software
NIH ImageJ software 1.47 for Windows (http://rsb.info.nih.gov/ij/)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Martin, M. V., Fiol, D. F., Zabaleta, E. J. and Pagnussat, G. C. (2014). Arabidopsis thaliana Embryo Sac Mitochondrial Membrane Potential Stain. Bio-protocol 4(10): e1128. DOI: 10.21769/BioProtoc.1128.
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Category
Plant Science > Plant cell biology > Cell imaging
Plant Science > Plant developmental biology > General
Cell Biology > Cell imaging > Confocal microscopy
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1,129 | https://bio-protocol.org/exchange/protocoldetail?id=1129&type=0 | # Bio-Protocol Content
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Karyotype Analysis
C Chun-Hung Chou
Muh-Hwa Yang
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1129 Views: 28156
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Jan 2013
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Jan 2013
Abstract
A chromosome is the structure that organizes DNA and protein in cells. It is a single piece of coiled DNA containing coding and non-coding sequences. Human cells have 23 pairs of chromosomes including 22 pairs of autosomes and one pair of sex chromosome, giving a total of 46 per cell. In tumor cells, chromosomal instability has been considered to be one of the hallmarks of tumor formation. Here we use the karyotype analysis to separate the chromosomes and observe the chromosomes in tumor cells with a microscope.
Keywords: Chromosome Chromosomal instability Tumor formation
Materials and Reagents
Cell lines: FaDu (ATCC, catalog number: HTB-43TM )
Trypsine (Life Technologies, Gibco®, catalog number: 15400-054 )
10% fetal bovine serum (Thermo Fisher Scientific, catalog number: SH30071.03 )
1% Penicillin-Streptomycin (10,000 U/ml; 10,000 μg/ml) (Life Technologies, Gibco®, catalog number: 15140-122 )
Colcemid (10 μg/ml) (Life Technologies, Gibco®, catalog number: 15212-012 )
Hypotonic solution (0.075 M KCl) (J.T.Baker®, catalog number: 7447-40-7 )
Glacial acetic acid (J.T.Baker®, catalog number: 64-19-7 )
Fixative (see Recipes)
Growth medium (see Recipes)
Equipment
10 cm cell culture dishes (Trueline Valve Corpotation, catalog number: TR4002 )
Centrifuge tube
Microscope (OLYMPUS, model: DP71 )
CO2 incubator
Centrifuge at room temperature (using “relative centrifugal force, rcf”)
Water bath
Procedure
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Category
Cancer Biology > Genome instability & mutation > Cell biology assays
Cell Biology > Cell imaging > Fixed-cell imaging
Cell Biology > Cell structure > Chromosome
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1,130 | https://bio-protocol.org/exchange/protocoldetail?id=1130&type=0 | # Bio-Protocol Content
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IFN-α/β Detection Assay Using Sensor Cell Lines
Valeria Lulla
Andres Merits
Aleksei Lulla
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1130 Views: 11237
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
Type I interferons (IFN-α/β) play an important role in host resistance to viral infections. Signaling through the JAK-STAT pathway, IFN-α/β stimulates response elements (ISRE) in the promoters of ISG to regulate their expression (reviewed in Reference 2). This method was adapted from InvivoGen to specifically detect and quantify IFN-α/β secreted in response to virus infection. HEK-Blue™ IFN-α/β cells were generated by stably introducing the human STAT2 and IRF9 genes into HEK293 cells to obtain a fully active type I IFN signaling pathway. The activation of this pathway is made detectable by the addition of a reporter gene expressing a secreted embryonic alkaline phosphatase (SEAP) under the control of the ISG54 promoter. ISG54 is a well-known ISG activated through an ISRE-dependent mechanism by type I IFNs.
Keywords: Interferon detection Virus Infection Sensor cell line Interferon response
Materials and Reagents
HEK-BlueTM IFN-α/β cells (InvivoGen, catalog number: hkb-ifnab )
QUANTI-Blue (InvivoGen, catalog number: rep-qb2 )
IFN-α2 (PBL Biomedical Laboratories, catalog number: PBL 11105-1 )
Dulbecco’s Modified Eagle’s Medium (DMEM) (PAA Laboratories GmbH)
Fetal Calf Serum (FCS) (Biochrom)
PBS (Naxo OÜ)
Trypsin/EDTA (GE Healthcare)
100x Penicillin-Streptomycin (Naxo OÜ)
Normocin (InvivoGen, catalog number: ant-nr-1 )
Zeocin (InvivoGen, catalog number: ant-zn-1 )
Blasticidin (Sigma-Aldrich, catalog number: 3513-03-9 )
Equipment
96-well plate
37 °C, 5% CO2 cell culture incubator
Microscope
UV cross-linker (Hoefer, model: UVC5000 )
Tecan SunriseTM microplate reader
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lulla, V., Merits, A. and Lulla, A. (2014). IFN-α/β Detection Assay Using Sensor Cell Lines. Bio-protocol 4(10): e1130. DOI: 10.21769/BioProtoc.1130.
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Category
Microbiology > Microbe-host interactions > Virus
Biochemistry > Protein > Expression
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1,131 | https://bio-protocol.org/exchange/protocoldetail?id=1131&type=0 | # Bio-Protocol Content
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Immunofluorescence Labeling of Pollen Tubes
HW Hao Wang
LJ Liwen Jiang
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1131 Views: 10895
Edited by: Tie Liu
Reviewed by: Samik Bhattacharya
Original Research Article:
The authors used this protocol in Nov 2013
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Abstract
Pollen tube is regarded as an excellent single-cell model system in plant cell studies. This protocol describes the use of a rapid and reliable immunofluorescence labeling method for studying in situ localization of proteins in pollen tubes. The whole experiment contains two major steps: pollen tube in vitro germination, and pollen tube fixation and immunolabeling. It takes about 2 days from pollen tube germination to immunofluorescence detection.
Materials and Reagents
Lily, tobacco or Arabidopsis pollen tubes
Note: Generally, mature lily and tobacco pollen grains are harvested 1-2 days after they are completely released from the anthers of flowers (Wang et al., 2010). For Arabidopsis, day-0 flowers are used for pollen collection (Boavida and McCormick, 2007).
1% glutaraldehyde
0.1% pectinase (Worthington Biochemical, catalog number: LS004298 )
1% cellulose (EMD Millipore, catalog number: 219466 )
1x phosphate buffered saline (PBS)
0.1% NaBH4 (INTERNATIONAL LABORATORY USA, catalog number: 1528397 )
Primary antibody
Secondary antibody
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A-7906 )
Sodium azide (Sigma-Aldrich, catalog number: S-2002 )
NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: I-8896 )
Gelatin (Sigma-Aldrich, catalog number: 48723-F )
Blocking buffer 1 (see Recipes)
Blocking buffer 2 (see Recipes)
Pollen species-specific germination media and culture conditions (see Recipes)
Equipment
Centrifuge with free angle rotor (Eppendorf, model: 5810R )
0.22 μm syringe filter (Merck Millipore, catalog number: SLGV033NS )
Confocal microscope (Leica Microsystems, model: TCS SP8 )
Incubator
Confocal dish (cover glass-bottom dish) (Life Scientific, catalog number: SPL-100350 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wang, H. and Jiang, L. (2014). Immunofluorescence Labeling of Pollen Tubes. Bio-protocol 4(10): e1131. DOI: 10.21769/BioProtoc.1131.
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Category
Plant Science > Plant cell biology > Cell staining
Cell Biology > Cell imaging > Fixed-tissue imaging
Cell Biology > Cell imaging > Fluorescence
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1,132 | https://bio-protocol.org/exchange/protocoldetail?id=1132&type=0 | # Bio-Protocol Content
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Purification and TEM of Afp and Its Variants
DR Daria Rybakova
AM Alok K. Mitra
MH Mark R. H. Hurst
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1132 Views: 9779
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
The Serratia entomophila antifeeding prophage (Afp), forms a phage-tail-like particle (tailocin) that causes cessation of feeding activity of the New Zealand grass grub, Costelytra zealandica. Here, we describe the more detailed purification protocol for Afp particles and its variants which is based on the procedure described in our original publication (Rybakova et al., 2013). The purification procedure includes inducing Escherichia coli (E. coli) cells harbouring afp genes under arabinose-inducible promoter for 24 h. The cells are harvested and sonicated on ice followed by DNAse treatment and centrifugation. The supernatant is then filter sterilised and applied to the size exclusion chromotography (SEC) column. The fractions containing Afp or its variants are pooled and ultracentrifuged. The supernatant is removed and the transparent pellet is resuspended in the residual buffer. The procedure results in Afp particles or variants thereof that are approximately 70% pure. The Afp particles are negatively stained and visualized using Transmission electron microscopy (TEM).
Materials and Reagents
Note: All solutions, unless otherwise specified, were prepared with distilled deionised Millipore-microfiltered (MilliQ) water, autoclaved for 15 min at 1.1 kg/cm2 at 121 °C and stored at room temperature. If antibiotics or supplements were to be added, the medium was cooled to a temperature of 50-60 °C before addition of antibiotic or supplement solution. The following antibiotics were used at stated concentrations (μg/ml): ampicillin, 100; chloramphenicol, 30; and spectinomycin, 100.
E. coli strains containing afp genes under arabinose promoter
Note: Please refer to the original manuscript (Rybakova et al., 2013) for strains and antibiotic resistance description.
LB-agar (Merck KGaA, catalog number: VM459383236 )
DNase (Roche Diagnostics, catalog number: 04716728001 )
0.7 % filtered (pore size 0.2 um) uranyl acetate diluted in sterile water (pH 5.0) (Electron Microscopy Sciences, catalog number: 22400 )
Sephacryl S-400 HR resin (GE, http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17395/17060901)
20% L-Arabinose (Acros organics) (Thermo Fisher Scientific, catalog number: AC104985000 ) (see Recipes)
0.4x Luria-Bertani broth (LB) (Life Technologies, catalog number: 12795-027 ) (see Recipes)
0. 2% sodium azide (Sigma-Aldrich, catalog number: S8032 ) (see Recipes)
250 mM TBS buffer (see Recipes)
TM buffer (see Recipes)
Equipment
Spectrophotometer
Orbital mixer incubator (Raytek Corporation)
1.7 ml microcentrifuge tubes
Eppendorf centrifuges
Sanyo soniprep 150 sonicator (18 Ω) (Panasonic Corporation)
0.45-µm-pore-size cellulose acetate filters (Advantec MFS, catalog number: 03CP045AN )
50 ml BD Falcon Tubes
Sephacryl S-400 HR beads (GE Healthcare, catalog number: 17-0609-10 ) for size exclusion chromatography (SEC)
The SEC column with a bed volume of 1.5 by 46 cm (Bio-Rad Laboratories, model: 737-4155 )
1.5 x 50 cm (D x L) column (Farmacia)
BioLogic LP system (Bio-Rad Laboratories, model: 731-8350 )
Beckman coulter Optima TM L-100K ultracentrifuge (Beckman Coulter, model: 392050 )
Polaron E5100 SEM coating unit
Note: This unit is from before 2000 and is no longer available, however a similar unit that is equivalent can be found at http://www.quorumtech.com/products/glow-discharge-system-free-standing.html.
Whatman #1 filter paper (Whatman)
Plastic-coated 200-mesh grid (ProSciTech, catalog number: GCU200 )
Morgagni 268D TEM (FEI)
Megapixel III digital camera
Note: The catalog numbers and model numbers are not important for reproducibility.
Polypropylen tubes used for the size exclusion chromotography (polypropylene 12 x 75 mm, 5 ml) (Labserv, catalog number: LBS504N )
Polypropylen tubes for ultracentrifugation (polycarbonate centrifuge bottles with cap assembly 16 x 76 mm, capacity 10.4 ml) (Beckman Coulter, catalog number: 355603 )
Falcon tubes (Thermo Fisher Scientific, catalog number: 14-432-22 )
Protect microorganism preservation system (Technical Service Consultants) for preservation of E. coli cells at -80 °C
Note: When required, a single inoculated bead was removed from the vial and dropped into a LB broth containing the appropriate antibiotic.
Procedure
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Category
Microbiology > Microbial biochemistry > Protein
Microbiology > Microbial cell biology > Cell imaging
Cell Biology > Cell imaging > Electron microscopy
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1,133 | https://bio-protocol.org/exchange/protocoldetail?id=1133&type=0 | # Bio-Protocol Content
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Barrier Function Assay
TD Tia DiTommaso
Floris Foijer
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1133 Views: 10577
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Feb 2013
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Abstract
This protocol serves to determine the integrity of the barrier function of (murine) epidermis. Defects in the barrier function lead to dehydration and infection in neonatal mice/humans. One possible way to assess epidermal barrier integrity is by a dye penetration assay as described hereunder. This assay should be done on unfixed, untreated tissues (e.g. formaldehyde- or glutaraldehyde-fixed) or on whole mouse embryos (E18.5). This protocol was adapted from Hardman (1998).
Keywords: Skin integrity Permiability Dye penetration assay
Materials and Reagents
E18.5 embryos from mice
Chilled methanol in water (25%, 50%, 75% and 100% MetOH)
0.1% toluidine blue (in water) (Sigma-Aldrich)
Chilled 1x PBS (pH 7.4) (Sigma-Aldrich, catalog number: P4417 ) (see Recipes)
Equipment
Forceps
Camera
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：DiTommaso, T. and Foijer, F. (2014). Barrier Function Assay. Bio-protocol 4(10): e1133. DOI: 10.21769/BioProtoc.1133.
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Category
Developmental Biology > Morphogenesis
Cell Biology > Tissue analysis > Tissue isolation
Cell Biology > Tissue analysis > Tissue staining
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1,134 | https://bio-protocol.org/exchange/protocoldetail?id=1134&type=0 | # Bio-Protocol Content
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Purification of HCV-remodeled and Control ER Membranes
David Paul
RB Ralf Bartenschlager
Published: Vol 4, Iss 10, May 20, 2014
DOI: 10.21769/BioProtoc.1134 Views: 7782
Original Research Article:
The authors used this protocol in Oct 2013
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Oct 2013
Abstract
As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate HCV-remodeled host cell membranes and endoplasmic reticulum (ER) membranes of naïve cells, by using a functional NS4B HA-tagged subgenomic replicon and a C-terminally HA-tagged calnexin-overexpressing cell line, respectively. Post-nuclear whole cell membrane fractions are first enriched by density gradient centrifugation, followed by HA-specific affinity tag purification. Upon elution under native conditions, purified samples can be subject to a variety of biochemical and functional assays.
Keywords: Hepatitis c virus Double membrane vesicles Affinity purification Plus-strand RNA virus Replication factory
Materials and Reagents
Huh7 human hepatoma cells, stably replicating a wt JFH1 subgenomic replicon (NS4Bwt) and a recombinant replicon cell line harboring the HA epitope and the adaptive mutation Q31R in NS4B (NS4BHA) (Paul et al., 2013) and Huh7 hepatoma cells stably overexpressing C-terminally HA-tagged calnexin (CANXHA) (Paul et al., 2013)
100 U/ml Pencillin and Streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
10% (v/v) Fetal Calf Serum (Life Technologies, Gibco®, catalog number: 10270-106 )
1x nonessential amino acids (Life Technologies, catalog number: 11140-035 )
Geneticin (Life Technologies, Gibco®, catalog number: 11811-098 )
Blasticidin (Life Technologies, Gibco®, catalog number: R210-01 )
Bradford reagent
NaN3 (Sigma-Aldrich, catalog number: S2002 )
Hemagglutinin peptide (Sigma-Aldrich, catalog number: I2149 )
Anti-HA agarose beads (Sigma-Aldrich, catalog number: A2095 )
1x protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 ) (see Recipes)
Bovine serum albumin (BSA) standard (see Recipes)
PBS (sterile) (see Recipes)
Hypotonic buffer (see Recipes)
Sucrose (see Recipes)
5 M NaCl solution (see Recipes)
IP washing buffer I (see Recipes)
IP washing buffer II (see Recipes)
IP elution buffer (see Recipes)
Equipment
15 cm cell culture dishes
37 °C, 5% CO2 cell culture incubator
Cell scraper (e.g. SARSTEDT AG, catalog number: 83.1830 )
Dounce homogenizer (1 ml) (e.g. WHEATON, catalog number: 357538 )
Table top cooling centrifuge with swinging buckets for 15 ml and 50 ml falcon tubes (e.g. Heraeus Multifuge 3-SR)
Open top polyclear ultracentrifuge tubes for SW40 rotor (Seton Identification Products, catalog number: 7031 )
Ultracentrifuge and SW40 rotor and buckets (e.g. Beckman Optima LE80K Ultracentrifuge and Beckman SW40Ti rotor and buckets)
Lab stand and clamp
19 G needle (e.g. BD, catalog number: 301500 )
Spectrophotometer (e.g. Amersham Ultrospec 2100 pro) and cuvettes (e.g. SARSTEDT AG, catalog number: 67.742 )
Rotator wheel for Eppendorf tubes (e.g. Labinco, model: L28, catalog number: 2800 )
Table top cooling centrifuge for Eppendorf reaction tubes (e.g. Eppendorf, model: 5417R )
Eppendorf Thermomixer compact
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Paul, D. and Bartenschlager, R. (2014). Purification of HCV-remodeled and Control ER Membranes. Bio-protocol 4(10): e1134. DOI: 10.21769/BioProtoc.1134.
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Category
Microbiology > Microbe-host interactions > Virus
Cell Biology > Organelle isolation > Membrane
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1,135 | https://bio-protocol.org/exchange/protocoldetail?id=1135&type=0 | # Bio-Protocol Content
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Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay
IL Isabelle S Lucet
JH Joanne M. Hildebrand
PC Peter E. Czabotar
Jian-Guo Zhang
NN Nicos A. Nicola
JS John Silke
JB Jeffrey J. Babon
JM James M. Murphy
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1135 Views: 12656
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
This protocol describes a robust technique for the measurement of pseudokinase-ligand interaction by a fluorescence-based Thermal Shift Assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulators of signal transduction and may therefore represent a novel class of drug targets. Unlike kinases, the catalytic efficiency of pseudokinases is rather poor or non existent, making it difficult to dissect the function of their nucleotide binding sites. Thermal denaturation-based methods have proven to be a powerful method for determining ligand binding capacity to purified pseudokinases and can inform on the potential drugability of the nucleotide binding site. This assay takes advantage of a change in flurorescence arising when a flurorescence dye, in this instance SYPRO® Orange, binds to hydrohobic patches that become exposed when a protein undergoes thermal denaturation. Ligand binding to a protein is known to increase its thermal stability which is reflected by a shift observed in the thermal denaturation curve between the unliganded protein and the liganded protein. This generalized protocol can also be tailored to other protein families. In addition, thermal denaturation-based methods can be used to identify optimal buffer conditions that may increase protein stability.
Materials and Reagents
Purified protein (stock solution preferably at a concentration above 20 μM) (Murphy et al., 2013)
1 M Dithiothreitol (DTT) stock (Astral Scientific, catalog number: C-1029 )
DMSO (high grade) (Sigma-Aldrich, catalog number: D-1435 )
MilliQ water
Nucleotide solutions (10 mM stock prepared in 20 mM Tris) (pH 8)
ATP (Sigma-Aldrich, catalog number: A2383 )
ADP (Sigma-Aldrich, catalog number: A2754 )
AMP-PNP (Sigma-Aldrich, catalog number: A2647 )
GTP (Sigma-Aldrich, catalog number: G8877 )
Divalent cations salt solutions (50 mM stock in MilliQ water)
MnCl2 (Sigma-Aldrich, catalog number: 203734 )
MgCl2 (Sigma-Aldrich, catalog number: M8266 )
Kinase inhibitor solutions (2 mM stock in 100% DMSO) (such as the pan-kinase inhibitors, Staurosporine, Sigma-Aldrich, catalog number: S5921 )
Thermal Shift Assay buffer (see Recipes)
SYPRO® Orange (Sigma-Aldrich, catalog number: S5692 ) (see Recipes)
Equipment
RT-PCR tubes GST-RG01 (Gene Targets Solutions)
1.5 ml microfuge tube
Qiagen/Corbett Rotor-Gene® 3000 RT-PCR machine (QIAGEN) (Murphy et al., 2013)
Software
Microsoft Excel or Prism
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lucet, I. S., Hildebrand, J. M., Czabotar, P. E., Zhang, J., Nicola, N. A., Silke, J., Babon, J. J. and Murphy, J. M. (2014). Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay. Bio-protocol 4(11): e1135. DOI: 10.21769/BioProtoc.1135.
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Category
Biochemistry > Protein > Fluorescence
Biochemistry > Protein > Interaction
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1,136 | https://bio-protocol.org/exchange/protocoldetail?id=1136&type=0 | # Bio-Protocol Content
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Preparation of Parasite Protein Extracts and Western Blot Analysis
A Arlett Heiber
TS Tobias Spielmann
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1136 Views: 24764
Edited by: Fanglian He
Reviewed by: Kanika Gera
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available to achieve this. If the protein is present within the parasite or is attached to a cellular structure of the iRBC cell, saponin can be used. This reagent lyses the membranes of infected and uninfected erythrocytes, the Maurer´s clefts (vesicular structures in the iRBC) and the parasitophorous vacuole membrane containing the parasite but leaves the parasite plasma membrane intact, providing a convenient procedure to isolate intact parasites without uRBCs. However, this method has the disadvantage that the host cell cytosol and the parasitophorous vacuole (PV) content of iRBCs are lost. If this has to be avoided, it is possible to use a Percoll gradient to separate intact iRBCs from uRBCs. Sequential treatment with Tetanolysin and saponin can then be used to selectively release the iRBC cytosol and the PV content from the parasite. These selective lysis methods are also suitable to determine the subcellular localisation of a protein of interest.
Keywords: Parasitology Malaria Plasmodium falciparum Western blotting
Materials and Reagents
Parasite culture
Plasmodium falciparum (e.g. clonal line 3D7)
Sterile, human 0+ erythrocyte concentrate (Blood bank)
RPMI complete medium (see Recipes)
RPMI-1640 (AppliChem GmbH, catalog number: A1538,9010 )
NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
Glucose (Merck KgaA, catalog number: 1.08342.1000 )
Albumax II (Life Technologies, Gibco®, catalog number: 11021-037 )
Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
40 mg/ml gentamicine (Ratiopharm)
Parasite protein extraction
Sorbitol (Sigma-Aldrich)
Triton X-114 (Enzo Life Sciences)
10x PBS (see Recipes)
0.03 % saponin lysis buffer (Sigma-Aldrich, catalog number: S4521 ) (see Recipes)
Parasite lysis buffer (see Recipes)
Tetanolysin (List Biological Labs, catalog number: 199 ) (see Recipes)
25x protease inhibitor cocktail mini (Roche Diagnostics, catalog number: 11836170001 ) (see Recipes)
Percoll solutions (GE Healthcare, catalog number: 17-0891-02 ) (see Recipes)
SDS-Page and western blot analysis
PageRuler Prestained Protein Ladder (Thermo Fisher Scientific)
Tris (Merck KGaA)
CAPS (Sigma-Aldrich)
SDS (SERVA Electrophoresis GmbH)
Low fat milk powder (blotting grade) (Carl Roth, catalog number: T145.2 )
ECL solution/Western Blot Detection Kit (Pierce Antibodies)
Antibodies (e.g. mouse anti-GFP, Roche Diagnostics, catalog number: 11814460001 ; horseradish peroxidase-conjugated goat anti-mouse, dianova GmbH, catalog number: 115-035-062 )
Electrophoresis buffer (see Recipes)
5x SDS sample buffer (see Recipes)
1 M Tris buffer (pH 6.8) (see Recipes)
1.5 M Tris buffer (pH 8.8) (see Recipes)
Polyacrylamide gel with 5% stacking gel and 12% separating gel (see Recipes)
CAPS buffer (1 L, 10 mM, pH 11.3) (see Recipes)
Blocking solution (50 ml) (see Recipes)
Equipment
Falcon tubes (15 ml, 50 ml)
Centrifuge
Eppendorf tubes (1.5 ml, 2 ml)
Sterilisation filters (0.22 µm)
Thermo block
Gel electrophoresis chamber (Bio-Rad Laboratories)
Nitrocellulose blotting membrane (Whatman, Protran®)
Chromatography paper (Grade 3 MM CHR) (GE Healthcare)
Tank blot device (Bio-Rad Laboratories)
Rolling device
Transparent sheets
Developer (Agfa-Gevaert Group)
Developing cassette
X-ray film (Agfa-Gevaert Group)
Procedure
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Copyright: © 2014 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:
Heiber, A. and Spielmann, T. (2014). Preparation of Parasite Protein Extracts and Western Blot Analysis. Bio-protocol 4(11): e1136. DOI: 10.21769/BioProtoc.1136.
Heiber, A., Kruse, F., Pick, C., Gruring, C., Flemming, S., Oberli, A., Schoeler, H., Retzlaff, S., Mesen-Ramirez, P., Hiss, J. A., Kadekoppala, M., Hecht, L., Holder, A. A., Gilberger, T. W. and Spielmann, T. (2013). Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog 9(8): e1003546.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Isolation and purification
Biochemistry > Protein > Immunodetection
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1,137 | https://bio-protocol.org/exchange/protocoldetail?id=1137&type=0 | # Bio-Protocol Content
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Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites
AH Arlett Heiber
SR Silke Retzlaff
TS Tobias Spielmann
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1137 Views: 9293
Edited by: Fanglian He
Reviewed by: Kanika Gera
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could also be used to detect antigens within the parasite if the sample is appropriately fixed and permeabilised before addition of the relevant antibody. While access of the antibody will avoid the detection problems often seen with post-embedding methods, this procedure will produce comparably poorer morphology.
Keywords: Parasitology Malaria Plasmodium falciparum Electron Microscopy
Materials and Reagents
Parasite culture
Plasmodium falciparum wildtype 3D7 parasites transfected with your protein of interest tagged with GFP
Sterile, human 0+ erythrocyte concentrate (Blood Banking)
RPMI complete medium (see Recipes)
RPMI-1640 (AppliChem GmbH, catalog number: A1538,9010 )
NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
Glucose (Merck KgaA, catalog number: 1.08342.1000 )
Albumax II (Life Technologies, Gibco®, catalog number: 11021-037 )
Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
Gentamicin (Ratiopharm GmbH)
Pre-embedding samples
Percoll (GE Healthcare, catalog number: 17-0891-02 )
Formaldehyde (Polysciences)
Tetanolysin (List Biological Labs, catalog number: 199 )
BSA (Enzo Life Sciences, Biomol®)
Mouse anti-GFP (Roche Diagnostics, catalog number: 11814460001 )
Rabbit anti-mouse linker antibodies (Dako, catalog number: Z0259 )
Protein A-Gold (6 nm) (Aurion, catalog number: 806-111 )
25% Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16210 )
OsO4 (Electron Microscopy Sciences, catalog number: 19152 )
Low melting agarose (Electron Microscopy Sciences, catalog number: 10207 )
100% ethanol (Merck KGaA)
Propylene oxide (Science Services GmbH, catalog number: UN1280 )
Gelantine capsules (Plano, catalog number: G29212 )
Uranyl acetate (Agar Scientific, catalog number: R1260 )
Methyl nadic anhydride (MNA) (Agar Scientific, catalog number: R1083 )
Dodecenylsuccinic anhydride (DDSA) (Agar Scientific, catalog number: R1053 )
EPON (Agar Scientific, catalog number: R1045 )
Benzyldimethylamine (BDMA) (Agar Scientific, catalog number: R1062b )
Pb(NO3)2 (Merck KGaA, catalog number: 12438 )
Na(CH3)2AsO2.3H2O (Fluca)
Saccharose (Merck KGaA)
10x PBS (see Recipes)
Tetanolysin (see Recipes)
Sodium cacodylate buffer (see Recipes)
EPON (see Recipes)
Lead citrate (pH 12) (Reynolds' stain) (see Recipes)
Equipment
Falcon tubes (15 ml, 50 ml)
Centrifuge
Eppendorf tubes (1.5 ml, 2 ml)
Sterilisation filters (0.22 µm)
Bunsen burner
Water bath
Glass bottles
Thermo block
Rolling device
Razor blades
Sealable glass vials
Tweezers
Ultramicrotome (Leica Microsystems)
Copper grids (Plano)
Transmission electron microscope (FEI)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial cell biology > Cell imaging
Biochemistry > Protein > Immunodetection
Cell Biology > Cell imaging > Electron microscopy
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1,138 | https://bio-protocol.org/exchange/protocoldetail?id=1138&type=0 | # Bio-Protocol Content
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Small-scale Subcellular Fractionation with Sucrose Step Gradient
YT Yuzuru Taguchi
Hermann M. Schätzl
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1138 Views: 16389
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Jun 2013
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Abstract
Here, we introduce the protocol for small-scale and simple subcellular fractionation used in our recent publication (Taguchi et al., 2013), which uses homogenization by passing through needles and sucrose step-gradient.
Subcellular fractionation is a very useful technique but usually a large number of cells are required. Because we needed subcellular fractionation of transiently-transfected cells, we developed a protocol for smaller numbers of cells. Our protocol for the subcellular fractionation is based on the protocol published by de Araújo and Huber (de Araujo et al., 2007), although substantial modifications have been made according to our experiences and information from personal communications. As optimal conditions seem to vary between cell lines, we advise to further modify the protocol to optimize for individual experiments. Our method is simple but sufficient for analysis of integral membrane proteins or proteins anchored to organelles by glycosylphosphatidylinositol or other lipid anchors, e.g. prion protein. However, proteins non-covalently attached to membranes or membrane proteins of organelles seem to be more prone to dissociation from the organelles during preparation and, if these proteins are the object of study, further modifications might be necessary.
Unlike in a continuous gradient, where a protein of interest is scattered over a wide range, step-gradient fractionation is advantageous in detection of relatively small amounts of proteins from small-scale experiments, because it concentrates the protein of interest in one fraction, if an appropriate combination of sucrose concentrations is used.
Keywords: Subcellular fractionation Sucrose step gradient Integral membrane protein GPI-anchored protein Prion protein
Materials and Reagents
Neuro2a cells (N2a)
Sucrose (Sigma-Aldrich, catalog number: S9378-500G )
1 M Tris-HCl (pH 7.1)
0.5 M EDTA (Millipore, catalog number: 324503-1KG )
Purified water
100 mg/ml solution of cycloheximide in DMSO (Sigma-Aldrich, catalog number: C4859-1ML )
Complete protease-inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
OptiMEM I supplemented with 10% fetal bovine serum
Phosphate-buffered saline without calcium/magnesium (Ca/Mg) (Life Technologies, catalog number: 10010-023 )
Deoxycholic acid (Sigma-Aldrich, catalog number: D2510-100G )
Triton X-100 (Sigma-Aldrich, catalog number: 93443-100ML )
Sodiumdodecyl sulfate (Sigma-Aldrich, catalog number: L6026-50G )
Glycerol (Sigma-Aldrich, catalog number: G9012-500ML )
Phosphate-buffered 0.5% Triton X-100 (TX100)/0.5% deoxycholate (DOC) lysis buffer (see Recipes)
5x sample buffer (see Recipes)
Equipment
6-well plate
1 ml BD Luer-LokTM disposable syringe (BD, catalog number: 309628 )
25G ultra-thin-wall needle (Terumo Medical Corporation, catalog number: NN-2525R )
Cell scraper
Centrifuge tube (thinwall, Ultra-ClearTM, 5 ml, 13 x 51 mm) (Beckman Coulter, catalog number: 344057 )
Ultracentrifuge (Beckman Coulter, model: L8-80M )
Pre-chilled swing bucket rotor (Beckman Coulter, model: SW50.1 )
Portable refractometer (optional) (e.g. ATAGO, model: PAL-1 )
Inverted microscope
Procedure
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Copyright: © 2014 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:
Taguchi, Y. and Schätzl, H. M. (2014). Small-scale Subcellular Fractionation with Sucrose Step Gradient. Bio-protocol 4(11): e1138. DOI: 10.21769/BioProtoc.1138.
Taguchi, Y., Mistica, A. M., Kitamoto, T. and Schätzl, H. M. (2013). Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. PLoS Pathog 9(6): e1003466.
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Category
Cell Biology > Organelle isolation > Fractionation
Biochemistry > Protein > Isolation and purification
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1,139 | https://bio-protocol.org/exchange/protocoldetail?id=1139&type=0 | # Bio-Protocol Content
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Small-scale Triton X-114 Extraction of Hydrophobic Proteins
YT Yuzuru Taguchi
Hermann M. Schätzl
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1139 Views: 17408
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Jun 2013
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Abstract
Here we introduce a protocol for Triton X-114 extraction which we used in our recently-published paper (Taguchi et al., 2013). It is a versatile method to concentrate or partially purify hydrophobic proteins. The presented protocol is based on the protocol published by Bordier (Bordier, 1981) but more simplified and down-scaled for more small-scale and simpler use (Taguchi et al., 2013).
Triton X-114 (TX114) is a non-ionic detergent which has a relatively low clouding point at 22 °C and separates into detergent (Det) and aqueous (Aq) phase at temperatures above the clouding point. During phase separation, hydrophobic solutes in the TX114 solution are sequestered to the Det phase, while hydrophilic solutes are sequestered to the Aq phase. Utilizing this phenomenon, TX114 extraction is a very versatile technique to efficiently concentrate hydrophobic proteins, especially glycosylphosphatidylinositol (GPI)-anchored proteins like the prion protein (PrP), because they have substantial amounts of highly hydrophobic moieties. Besides, phase separation using TX114 tolerates a variety of conditions, e.g. different pH or relatively low concentrations of guanidine hydrochloride. Since the hydrophobic proteins are sequestered to the Det phase as long as the phase separation occurs, and if the hydrophobicity of the protein of interest is not affected by pH or denaturant, this technique can be also utilized to change buffers or to remove denaturants. When using enzymes or proteases which maintain activities in detergent solutions, TX114 can also be used to separate hydrophobic from the water-soluble hydrophilic moieties upon enzymatic digestion of proteins, as done by us using in vitro digestion of PrP with phosphatidylinositol-specific phospholipase C (Taguchi et al., 2013).
Keywords: Triton X-114 extraction Hydrophobic protein GPI-anchored protein Prion protein PIPLC digestion
Materials and Reagents
Neuro2a cells (N2a) (100%-confluent on a well of a 6-well plate)
Triton X-114 (Sigma-Aldrich, catalog number: X 114-100 ML )
10x phosphate-buffered saline (Sigma-Aldrich, catalog number: P5493-1L )
Complete protease-inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
Note: Optional, depending on the kind of the protein of interest and the purpose of experiments.
0.5 M EDTA (Millipore, catalog number: 324503-1KG )
Deoxycholic acid (Sigma-Aldrich, catalog number: D2510-100G )
Triton X-100 (Sigma-Aldrich, catalog number: T8787-100 ML )
Sodiumdodecyl sulfate (Sigma-Aldrich, catalog number: L6026-50G )
Glycerol (Sigma-Aldrich, catalog number: G9012-500ML )
Purified water
Phosphate-buffered saline without calcium and magnesium (Ca/Mg) (Life Technologies, catalog number: 10010-023 )
Phosphate-buffered 0.5% Triton X-100 (TX100)/0.5% deoxycholate (DOC) lysis buffer (see Recipes)
5x sample buffer (see Recipes)
Equipment
Refrigerated centrifuge (max rcf 16,100 x g) (Eppendorf, model: 5415R )
Non-refrigerated centrifuge (max rcf 21,130 x g) (Eppendorf, model: 5424 )
50 ml conical tube
6-well plate
1.5 ml plastic tube
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Biochemistry > Protein > Isolation and purification
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1,140 | https://bio-protocol.org/exchange/protocoldetail?id=1140&type=0 | # Bio-Protocol Content
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Novel Method for Site-specific Induction of Oxidative DNA Damage to Study Recruitment of Repair Proteins to Heterochromatin and Euchromatin
Leizhen Wei
Satoshi Nakajima
AL Arthur S. Levine
Li Lan
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1140 Views: 9328
Reviewed by: Fanglian HeClaudia Catalanotti
Original Research Article:
The authors used this protocol in Nov 2013
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Abstract
ROS-induced DNA damage is repaired in living cells within a temporal and spatial context, and chromatin structure is critical to a consideration of DNA repair processes in situ. It’s well known that chromatin remodeling factors participate in many DNA damage repair pathways, indicating the importance of chromatin remodeling in facilitating DNA damage repair. To date, there has been no method to induce site-specific oxidative DNA damage in living cells. Therefore, it is not known whether the DNA repair mechanisms differ within active or condensed chromatin. We recently established a novel method, DTG (Damage Targeted at one Genome-site), to study DNA damage response of reactive oxygen species (ROS)-induced DNA damage in living cell at one genome loci with active or inactive transcription. For this, we integrated a tetracycline responsive elements (TRE) cassette (~90 kb) at X-chromosome in U2OS cells (Lan et al., 2010), then fused KillerRed (KR), a light-stimulated ROS-inducer which can specifically produce ROS-induced DNA damage, to a tet-repressor (tetR-KR, OFF) or a transcription activator (TA-KR, ON) (Lan et al., 2014) (Figure 1). TetR-KR or TA-KR binds to the TRE cassette and induces ROS damage under hetero- or euchromatin states, respectively. How chromatin states regulate the DNA damage response processes can be examined by using this powerful method.
Figure 1. Scheme of the DTG system. A. Scheme of tetR and TA tagged KR expression in the U2OS TRE cell line. To induce ROS-mediated damage at a specific locus in the genome, we fused KR to the tetracycline repressor to induce ROS damage in a 90 kb tetracycline response element (TRE) array (totally 96 repeats) in U2OS cells. B. Expression of tetR-KR in U2OS TRE cell line.
Materials and Reagents
U2OS-SCE cell line (made in our laboratory) (Lan et al., 2010)
Notes:
In this cell line, 200 copies of pTRE / I-SceI were integrated in U2OS cells.
Cells are cultured with Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (EuroClone S.p.A. P.IVA, catalog number: ECM0103L ) with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F9665 ).
For preparation of cells, cells were washed with PBS without Ca2+ and Mg2+ (EuroClone S.p.A. P.IVA, catalog number: ECB4004L ), trypsinized with Trypsin/EDTA without out Ca2+ and Mg2+ (Thermo Fisher Scientific, catalog number: BW17161E ).
Plasmids
pBROAD3/tetR-KR
pBROAD3/tetR-mCherry
pBROAD3/TA-KR
pBROAD3/TA-mCherry
Note: pBROAD3/tetR-mcherry was provided by Dr. Edith Heard (Masui et al., 2011); pBROAD3/tetR-KR, pBROAD3/TA-mCherry, pBROAD3/TA-KR were made in our laboratory (Lan et al., 2014). Sequences are available based on requests.
GFP-fusion protein or endogenous protein
Lipofectamine 2000 (Life Technologies, catalog number: 12566-014 )
Opti-MEM (Life Technologies, catalog number: 51985-091 )
35 mm glass bottom culture dishes (MatTek, catalog number: P35GC-1.5-14-C )
IMMOIL-F30CC (Chip Humphries, catalog number: Z-81225 )
Equipment
35 mm glass bottom culture dishes
37 °C, 5% CO2 cell culture incubator
Olympus FV1000 confocal microscopy system (OLYMPUS, model: FV1000- FILTER DETECT ; SYS.PACKAGE: IX81-1 405/M_AR/559/635N) with 488, 559 nm lasers
PLAPON 60x oil lens (super chromatic abe. corr. obj W/1.4NA FV) (OLYMPUS, catalog number: FM1-U2B990 )
Thermo-plate (MATS-U52RA26 for IX81/71/51/70/50, metal insert, HQ control) (OLYMPUS, catalog number: OTH-I0126 )
15 watt cool white fluorescent bulb (OSRAM SYLVANIA)
A stage UVP (Upland, CA)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cell Biology > Cell structure > Chromosome
Molecular Biology > DNA > DNA damage and repair
Biochemistry > Other compound > Reactive oxygen species
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1,141 | https://bio-protocol.org/exchange/protocoldetail?id=1141&type=0 | # Bio-Protocol Content
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Identification of Helminth-induced Type 2 CD4+ T Cells and ILC2s
Mario M. Zaiss
KM Kendle M. Maslowski
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1141 Views: 8278
Reviewed by: Lee-Hwa Tai
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
After activation, T cells differentiate into different T helper (Th) subsets, namely Th1, Th2, and Th17. These different Th subsets are associated with the production of particular cytokines endowing them with different functions. In immunity against helminth infections the Th2 cell subset plays an important role. Th2 cells typically produce IL-4, IL-5, IL-13, and IL-9 resulting in antibody-isotype switching to IgE, eosinophilia, basophilia, mucin production, and smooth muscle cell hyperactivity. Here we analyze the development of the pathogen specific Th2 immune responses in mice after infection with the helminth parasite, Heligmosomoides polygyrus bakeri, the induction of innate lymphoid cells type 2 (ILC2) and the activation of the inflammasome in macrophages by excretory/secretory products of Heligmosomoides polygyrus bakeri.
Part I.Generation and collection of Heligmosomoides poylgyrus (Hp) excretory/secretory (ES) products (HES)
Materials and Reagents
Adult L5 Heligmosomoides poylgyrus (Hp)
50 ml PBS (Life Technologies, Gibco®, catalog number: 10010-015 )
Complete RPMI (GE Healthcare, catalog number: E15-840 )
10,000 MWCO cellulose membrane (EMD Millipore, Centriprep®)
EndoTrap Blue 5/1 LPS-binding affinity column (Hyglos GmbH, catalog number: 311063 )
HES wash solution I (see Recipes)
HES wash solution II (see Recipes)
HES culturing solution (see Recipes)
Equipment
Aluminum foil
50 ml Falcon tube (BD, catalog number: 352070 )
Pipette
Falcon flask (TPP Techno Plastic Products, catalog number: 90076 )
Nanodrop (Thermo Fisher Scientific)
Centrifuge (Eppendorf, model: 5810R )
Perti dish
Microscope (OLYMPUS, model: CKX31 )
Laminar airflow cabinet (Faster Ultrasafe, model: US218D )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Immune cell function > Cytokine
Immunology > Host defense > Murine
Immunology > Immune cell function > Lymphocyte
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1,142 | https://bio-protocol.org/exchange/protocoldetail?id=1142&type=0 | # Bio-Protocol Content
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In vitro Inflammasome Assay
Mario M. Zaiss
KM Kendle M. Maslowski
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1142 Views: 13545
Reviewed by: Lee-Hwa Tai
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
Innate immune cells sense pathogen and danger-associated molecular patterns (PAMPs and DAMPs) through a range of innate immune pattern recognition receptors (PRRs). One type of PRRs are the Nod-like receptors (NLRs), which form inflammasomes; a molecular platform required for the recruitment and activation of Caspase-1, which in turn cleaves and activates IL-1β, IL-18. Examples of inflammasome forming NLRs are NLRP3, NLRP1, NAIP and NLRC4. We can easily identify new inflammasome activators by performing the following protocol.
Materials and Reagents
Mice (e.g. C57B/6)
DMEM (Life Technologies, Gibco®, catalog number: 10566-024 )
1% Penicillin-Streptomycin (10,000 U/ml) (Life Technologies, Gibco®, catalog number: 15140148 )
1x PBS
Accutase (PAA Laboratories GmbH, catalog number: L11-007 )
Ultrapure Escherichia coli (E.coli) K12 LPS (Life Technologies, InvitrogenTM, catalog number: tlrl-peklps )
IL-1β ELISA (eBioscience, catalog number: 88-7013-88 )
Primary antibodies against IL-1β (e.g. R&D System, catalog number: AF-401-NA ) and Caspase-1 (e.g. Aidpogen International, catalog number: AG-20B-0042-C100 ) (suitable for western blot)
HRP-conjugated secondary antibody (e.g. Cell Signaling anti-mouse HRP, Cell Signaling Technology, catalog number: 7076 )
Nitrocellulose membrane (0.45 µm) (GE Healthcare, Hybond, catalog number: 95038-402 )
5% sodium azide in water (Sigma-Aldrich, catalog number: 26628-22-8 )
Skim milk powder (Sigma-Aldrich, or your local grocer)
ECL solution (Pierce, catalog number: 34095 or GE Healthcare, catalog number: RPN2133 )
1 M dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
Stimulus: e.g. 1 M ATP (Sigma-Aldrich, catalog number: A26209 ), 10 mM nigericin (Sigma-Aldrich, catalog number: N7143 ), 300 µg/ml monosodium urate (Michigan State University, catalog number: U2875 )
Flushing medium (see Recipes)
Red blood cell lysis solution (see Recipes)
Bone-marrow-derived macrophage (BMM) culture medium (see Recipes)
Ponceau staining solution (see Recipes)
3x western blot sample buffer (see Recipes)
Blocking buffer (see Recipes)
Running buffer (see Recipes)
Blotting buffer (see Recipes)
Equipment
Razor blade
Laminar flow hood
Bench top centrifuge with 96-well plate adaptors
10 ml syringe
22 G gauge needles
18 G blunt needles
100-µm cells strainers
10 cm cell-culture treated petri dishes
MaxiSorb ELISA plates (Nunc®)
96-well tissue culture plate
ELISA plate reader
Western blot equipment (protean mini 1 mm) (Bio-Rad Laboratories)
Film, developer, dark room or equivalent development equipment
Parafilm or plastic
Software
ELISA analysis software
Procedure
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Copyright: © 2014 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:
Zaiss, M. M. and Maslowski, K. M. (2014). In vitro Inflammasome Assay. Bio-protocol 4(11): e1142. DOI: 10.21769/BioProtoc.1142.
Zaiss, M. M., Maslowski, K. M., Mosconi, I., Guenat, N., Marsland, B. J. and Harris, N. L. (2013). IL-1beta suppresses innate IL-25 and IL-33 production and maintains helminth chronicity. PLoS Pathog 9(8): e1003531.
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Category
Immunology > Immune cell function > Cytokine
Biochemistry > Protein > Immunodetection
Immunology > Immune cell isolation > Pattern recognition receptor
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1,143 | https://bio-protocol.org/exchange/protocoldetail?id=1143&type=0 | # Bio-Protocol Content
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Protocol for Preparation of Nuclear Protein from Mouse Lungs
ZX Zuoxiang Xiao
QJ Qun Jiang
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1143 Views: 13895
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Apr 2013
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Abstract
This protocol describes how to extract nuclear protein from mouse lungs, including tissue preparation, stepwise lysis of cells and centrifugal isolation of nuclear protein fraction. This is an efficient method to get comparable nuclear protein extracts from lung tissues.
Materials and Reagents
Mice
DNase I (100 mg/ml) (Sigma-Aldrich, catalog number: DN25 )
1x PBS
HEPES (Life Technologies, Gibco®, catalog number: 15630-080 )
Magnesium chloride solution (Sigma-Aldrich, catalog number: M1028 )
Potassium chloride solution (Sigma-Aldrich, catalog number: 60121 )
Nonidet P40 (NP-40) (Roche Diagnostics, catalog number: 11332473001 )
Fetal calf serum (FCS)
Bovine serum albumin (BSA)
0.25% trypsin (Life Technologies, Gibco®, catalog number: 15050 )
CD45 MicroBeads kit (Miltenyi Biotec, catalog number: 130-052-301 )
Cytoplasmic extract buffer (see Recipes)
Nuclear extract buffer (see Recipes)
Equipment
Tweezers and scissors
26 G1/2 needles (BD, catalog number: 309659 )
Cell strainer (100 μm and 40 μm) (BD Biosciences, Falcon®, catalog numbers: 352360 and 352340 )
10 ml and 1 ml syringe (BD, catalog numbers: 309604 and 305111 )
Filter tips (Eppendorf)
15 ml Corning tubes (Corning)
DNase, RNase-free Eppendorf tubes (Eppendorf)
Tissue culture dishes (60 x 15 mm style) (BD Biosciences, Falcon®, catalog number: 353002 )
MACS device (Miltenyi Biotec, model: 0 16210 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Xiao, Z. and Jiang, Q. (2014). Protocol for Preparation of Nuclear Protein from Mouse Lungs. Bio-protocol 4(11): e1143. DOI: 10.21769/BioProtoc.1143.
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Category
Biochemistry > Protein > Isolation and purification
Cell Biology > Organelle isolation > Nuclei
Systems Biology > Proteomics > Nuclear
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1,144 | https://bio-protocol.org/exchange/protocoldetail?id=1144&type=0 | # Bio-Protocol Content
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Protocol for Macrophage Depletion from Mice
ZX Zuoxiang Xiao
QJ Qun Jiang
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1144 Views: 18766
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Apr 2013
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Apr 2013
Abstract
Macrophage depletion has been used extensively to study autoimmune disease and more recently in tumor models. The clodronate-containing liposomes will be recognized as foreign particles and get engulfed by macrophages upon dosing into the animal by the chosen routes. Consequently, macrophages that have engulfed liposomes will all be destroyed by the liposomal. In the protocol presented here the clodronate-containing liposomes were used to systemically deplete macrophages in mice.
Materials and Reagents
Mice
Clodronate (dichloromethylene diphosphonic acid)-loaded liposomes (VUmc)
PBS-containing liposomes (VUmc)
Phosphatidyl choline (Lipoid GmbH, catalog number: D-67065)
Cholesterol (Sigma-Aldrich, catalog number: C8667 )
Liposomes (see Recipes)
Liposomes suspended in sterile phosphate buffered saline (see Recipes)
Equipment
Syringe (10 ml and 1 ml) (BD, catalog numbers: 309604 and 305111 )
26 G 1/2 needles (BD, catalog number: 309659 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Xiao, Z. and Jiang, Q. (2014). Protocol for Macrophage Depletion from Mice . Bio-protocol 4(11): e1144. DOI: 10.21769/BioProtoc.1144.
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Category
Immunology > Animal model > Mouse
Immunology > Immune cell function > Macrophage
Biochemistry > Lipid > Extracellular lipids
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1,145 | https://bio-protocol.org/exchange/protocoldetail?id=1145&type=0 | # Bio-Protocol Content
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Competitive Bone-marrow Transplantations
M Maria Maryanovich
AG Atan Gross
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1145 Views: 24855
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in May 2012
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May 2012
Abstract
Competitive bone marrow transplantation assay measures multi-lineage reconstitution of hematopoiesis in irradiated transplant recipient mice. Thus this assay is routinely used to determine haematopoietic stem and progenitor cells (HSPCs) functionality in vivo. The principle of the method is to transplant bone marrow donor cells (derived from transgenic mice of choice) on C57BL6 background together with normal competitor bone marrow. In order to distinguish donor from competitor cells upon transplantation, usually competitor mice are congenic and carry the differential B cell antigen originally designated Ly5.1 and CD45.1.
A typical competitive bone marrow transplantation experiment will contain two transplantation groups, donor (transgenic mice of choice and their controls) are transplanted in competition with normal competitors and engraftment efficiency is evaluated in both blood and bone marrow.
Keywords: Bone marrow transplantation Hematopoesis HSCs Competitive Stem cell engrafment
Materials and Reagents
C57BL6 (CD45.2) mice (bred in our laboratory)
C57BL6 (CD45.1) mice (bred in our laboratory)
Ciprofloxacin 200 (200 mg/100 ml Bayer)
Phosphate Buffered Saline (PBS) (without Ca2+ and Mg2+) (Sigma-Aldrich, catalog number: D8537 )
Turk’s solution (Merck KGaA, catalog number: 1.09277.0100 )
Heparin sodium salt (Sigma-Aldrich, catalog number: H3393-100KU )
Ficoll-Paque plus (GE Healthcare, catalog number: 17-1440-03 )
FITC anti-mouse CD45.1 (cloneA20) (Biolegend, catalog number: 110705 )
PE anti-mouse CD115 (clone AFS98) (Biolegend, catalog number: 135506 )
APC anti-mouse B220 (clone RA3-6B) (eBioscience, catalog number: 17-0552-81 )
FITC anti-mouse Gr-1 (clone RB6-8C5) (Biolegend, catalog number: 108405 )
APC anti-mouse Ter119 (clone TER-119) (Biolegend, catalog number: 116211 )
PE anti-mouse CD3 (clone 145- 2C11) (Biolegend, catalog number: 100307 )
APC anti-mouse NK1.1 (clone PK136) (Biolegend, catalog number: 108709 )
Streptavidin PerCp (Biolegend, catalog number: 405213 )
Sodium azide
FACS buffer (see Recipes)
RBC lysis buffer (see Recipes)
Heparin solution (see Recipes)
Equipment
3 ml syringe (BD Biosciences, catalog number: 305270 )
Forceps and sharp scissors
Needles (21 G x 1.5-Nr.2 0.8 mm x 40 mm) (BD Biosciences, catalog number: 304432 )
0.5 ml syringes (BD, catalog number: 320933 )
Mouse restrainer
Red light lamp
Tissue culture (TC) dish (6 well plate) (Corning, catalog number: 3516 )
15 ml conical tubes (BD Biosciences, Falcon®, catalog number: 352196 )
70 μM nylon strainers (BD Biosciences, Falcon®, catalog number: 352350 )
Inverted microscope
Ice bucket
Hemacytometer (Sigma-Aldrich, catalog number: Z359629-1EA )
Centrifuge
Scalpel knife
Eppendorf tubes (Axygen®, catalog number: MCT-175-C ).
FACS micro-tubes (USA Scientific, catalog number: 1412-1000 )
FACS tubes (BD, catalog number: 352054 )
Mice Irradiation machine (Gamma cell 40 Exactor) (InsideView)
BD LSR II flow cytometer
Software
FACSDiva software
FlowJo software
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Maryanovich, M. and Gross, A. (2014). Competitive Bone-marrow Transplantations. Bio-protocol 4(11): e1145. DOI: 10.21769/BioProtoc.1145.
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Category
Stem Cell > Adult stem cell > Hematopoietic stem cell
Immunology > Animal model > Mouse
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1,146 | https://bio-protocol.org/exchange/protocoldetail?id=1146&type=0 | # Bio-Protocol Content
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Peer-reviewed
Purification and Structural Analysis of QS-inhibiting Compounds from Staphylococcus delphini
YC Ya-Yun Chu
MN Mulugeta Nega
FG Friedrich Götz
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1146 Views: 7709
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Sep 2013
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Sep 2013
Abstract
The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we present a purification protocol for molecules excreted by a group of Gram-positive zoonotic pathogen bacteria, the ‘Staphylococcus intermedius group’, that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini (S. delphini). They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. These substances can be isolated and purified from the culture supernatant using this upscalable purification method.
Materials and Reagents
Staphylococcus delphini DSMZ20771 strain (DSM number: 20071 )
Note: DSMZ stands for Deutsche Sammlung von Mikroorganismen und Zellkulturen.
Tryptic Soy Broth (TSB) (Sigma-Aldrich, catalog number: T8907 )
Amberilite XAD-16 resin (Sigma-Aldrich, catalog number: 1-0379 )
Methanol
Acetic acid (Merck KGaA)
Amberilite IRC 50 cation exchange resin (SERVA Electrophoresis GmbH, catalog number: 40501 )
Sodium hydroxide
Ethanol
50 mM and 1 M sodium phosphate buffer
SP sepharose cation exchange column (GE Healthcare, catalog number: 17-5161-01 )
Sodium chloride
Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: T6508 )
Phosphoric acid for HPLC (Sigma-Aldrich, catalog number: 79606 )
Acetonitrile for HPLC (Mallinckrodt Baker, catalog number: 9012 )
Equipment
37 °C shaking incubator (Infors AG)
Centrifuge (Eppendorf)
Rotary evaporator (BÜCHI Labortechnik AG)
Äkta FPLC equipped with P-900, UV-900, PH/C-900 (GE Healthcare)
Preparative HPLC System equipped with Bischoff HPLC compact pump QC-P 2250 and Multiwavelength detector QC-1157 (Bischoff)
Nucleosil 100 C-18 (8 x 250 mm column) (MACHEREY-NAGEL, catalog number: 715332.80 )
Agilent 1200 series HPLC system (Agilent)
Waters XBridge C18 (5 mm, 4.6 x 150 mm column) (Waters, part number: 186003116 )
Procedure
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Copyright: © 2014 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:
Chu, Y., Nega, M. and Götz, F. (2014). Purification and Structural Analysis of QS-inhibiting Compounds from Staphylococcus delphini. Bio-protocol 4(11): e1146. DOI: 10.21769/BioProtoc.1146.
Chu, Y. Y., Nega, M., Wölfle, M., Plener, L., Grond, S., Jung, K. and Götz, F. (2013). A new class of quorum quenching molecules from Staphylococcus species affects communication and growth of gram-negative bacteria. PLoS Pathog 9(9): e1003654.
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Category
Microbiology > Microbial signaling > Quorum sensing
Microbiology > Microbial biochemistry > Other compound
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1,147 | https://bio-protocol.org/exchange/protocoldetail?id=1147&type=0 | # Bio-Protocol Content
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Infant Rabbit Colonization Competition Assays
Yang Fu
JM John J. Mekalanos
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1147 Views: 9939
Original Research Article:
The authors used this protocol in Dec 2013
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Abstract
Enteric pathogenic bacteria such as Vibrio cholerae and enteropathogenic Escherichia coli (E. coli) cause life-threatening diarrheal diseases that have afflicted humans for centuries. Understanding the effectors required for intestinal colonization is very important to research on bacteria pathogenesis, and is also important to testing new therapeutics and development of the novel vaccines. Here, we describe the Infant Rabbit Colonization Competition Assay, a variant method of the powerful, nonsurgical animal model reported by Ritchie et al. (2010). In our modified assay, wild type and mutant strains are mixed together and inoculated into 2-day-old New Zealand white rabbits. The competitive index for each mutant measures the colonization capacity of the mutant relative to its wild type parental strain in the gastrointestinal tract. Compared to the traditional Sucking Mice model, the clinical and histologic signs of Vibrio cholerae (V. cholerae)-induced disease of infant rabbits more closely resemble human cholera. The larger input bacteria amount of this model also facilitates high-throughput screens, such as Tn-Seq technology (Fu et al., 2013).
Keywords: Vibrio Cholerae Enteric Bacteria Animal Model Colonization Factor Competetion Assay
Materials and Reagents
New Zealand white infant rabbit (2-5 days old) (Millbrook farm)
Lactating doe (Millbrook farm)
Vibrio cholerae
Note: Alternatively, any enteric pathogenic bacteria that can successfully colonize the infant rabbit gut system can be used in this protocol.
Zantac (GlaxoSmithKline, NDC: 0173-0363-01 )
40 mEq/20 ml potassium chloride (KCl) (Hospira, NDC: 0409-6653-05 )
Isoflurane (USP) (Piramal Enterprises, NDC: 66794-013-25 )
Phosphate buffered saline (PBS) (Lonza, catalog number: 51225 )
X-gal and/or antibiotics (depend on the selective marker of the experimental strain)
70% ethanol
2.5% sodium bicarbonate buffer (pH 8.0) (see Recipes)
Luria-Bertani broth (LB) medium and LB agar solid medium (see Recipes)
Equipment
BL2 animal facility
Fume hood
37 °C incubator or warm room
1 ml sterile syringe (BD, catalog number: 305553 )
3 ml sterile syringe (BD, catalog number: DG508504 )
26 G 5/8 needle (BD, catalog number: 305115 )
Surgical scissors and tweezers (Fine Science Tools)
Centrifuge (Eppendorf, model: 5424 )
1.5 ml sterile microcentrifuge tube
96 wells tissue culture plate (BD, catalog number: 353072 )
Size 5 French catheter
Silk ligature
Shaker
Spectrophotometer
Mini-bead beater-16 (Bio Spec Products, catalog number: 607 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Fu, Y. and Mekalanos, J. J. (2014). Infant Rabbit Colonization Competition Assays. Bio-protocol 4(11): e1147. DOI: 10.21769/BioProtoc.1147.
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Category
Microbiology > Microbe-host interactions > In vivo model
Immunology > Host defense > General
Immunology > Animal model > Rabbit
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1,148 | https://bio-protocol.org/exchange/protocoldetail?id=1148&type=0 | # Bio-Protocol Content
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Intracellular Glycogen Assays
Yong Jun Goh
TK Todd R. Klaenhammer
Published: Vol 4, Iss 11, Jun 5, 2014
DOI: 10.21769/BioProtoc.1148 Views: 12615
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
Glycogen, a soluble multi-branched glucose homopolysaccharide, is composed of chains of α-1,4-linked glucose residues interconnected by α-1,6-linked branches. The classical biosynthetic pathway involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyltransferase (GlgC or GlgCD), glycogen synthase (GlgA) and branching enzyme (GlgB). Phosphoglucomutase converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC or GlgCD. Then, GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear α-1,4-glucan. GlgB subsequently cleaves off portions of the glucan and links it to internal glucose molecules in existing chains via α-1,6 glycosidic bonds to form the glycogen structure. Glycogen breakdown is mediated by glycogen phosphorylase (GlgP) and debranching enzyme (GlgX), which catalyze the sequential phosphorolysis of α-1,4-glucosyl linkages in the glucan chain from the non-reducing ends and debranching of the limit dextrins generated by GlgP, respectively. An increasing number of studies have revealed the involvement of glycogen metabolism in a multitude of physiological functions in some prokaryotes beyond the function of synthesizing energy storage compounds. Lactobacillus acidophilus NCFM was the first probiotic lactic acid bacterium demonstrated to possess a functional glycogen biosynthesis pathway that is involved in its growth, bile tolerance and complex carbohydrate metabolism (Goh and Klaenhammer, 2013). The following qualitative (for detection of intracellular glycogen) and quantitative (for measurement of intracellular glycogen content) intracellular glycogen assay protocols for Lactobacillus acidophilus (L. acidophilus) were modified from previous works (Govons et al., 1969; Law et al., 1995; Parrou and Francois, 1997) and should be applicable to other lactic acid bacteria as well as most microorganisms.
Part I. Qualitative detection of intracellular glycogen with iodine-staining method
Materials and Reagents
L. acidophilus strains, or desired bacterial strains
MRS broth (Difco), or other liquid growth medium of choice (stored at 4 °C)
Iodine solution (see Recipes)
Solid semi-defined medium (SDM) with 2% (w/v) carbohydrate (see Recipes)
Equipment
37 °C incubator
37 °C anaerobic chamber incubator
Pipettor
Procedure
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Category
Microbiology > Microbial metabolism > Carbohydrate
Biochemistry > Carbohydrate > Glycogen
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1,149 | https://bio-protocol.org/exchange/protocoldetail?id=1149&type=0 | # Bio-Protocol Content
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Protoplast Preparation and Determination of Cell Death
Antoine Danon
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1149 Views: 18181
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Oct 2013
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Abstract
The protoplasts assay constitutes a powerful tool that allows an easy uptake of active agents and a precise quantification of cell death induction in different populations. Our study showed that the basal level of cell death in our controls is low and stable throughout the length of our experiments (Danon et al., 2005; Pineau et al., 2013). In addition, the data obtained from the protoplast assay are applicable to intact seedlings, where it is possible to see differences in the intensity of necrotic lesions (Danon et al., 2006) even if those differences are not as easily and clearly quantifiable as with the protoplast assay.
Keywords: Protoplast Arabidopsis thaliana Cell death Evans blue
Materials and Reagents
Plant materials: Arabidopsis thaliana sterile seedlings grown in vitro from 1 to 3 weeks in the required conditions
Gamborg B5 medium salt and vitamins (Duchefa Biochemie, catalog number: G0210 )
2-(N-Morpholino) ethanesulfonic acid (MES)
Mannitol
Digestion enzymes
Cellulase (Duchefa Biochemie, catalog number: C8001 )
Macerozyme (Duchefa Biochemie, catalog number: M8002 )
Culture medium (see Recipes)
Digestion medium (see Recipes)
W5A medium (see Recipes)
21% sucrose (see Recipes)
1% Evans blue (see Recipes)
Equipment
Light microscope
An appropriate slide (e.g. Malassez slide)
Centrifuge for 15 ml and 50 ml Falcon tubes
Sterile pipette
Filter (0.45 µm)
Hemocytometer (VWR International, catalog number: 631-0975 )
Cell dissociation sieve (100 µm) (Sigma-Aldrich, catalog number: CD1-1KT ) sieve mounted on a sterile 150 ml beaker
Micropore tape (VWR International, catalog number: 115-8172 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Danon, A. (2014). Protoplast Preparation and Determination of Cell Death. Bio-protocol 4(12): e1149. DOI: 10.21769/BioProtoc.1149.
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Category
Plant Science > Plant cell biology > Cell isolation
Cell Biology > Cell viability > Cell death
Cell Biology > Cell staining > Whole cell
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1,150 | https://bio-protocol.org/exchange/protocoldetail?id=1150&type=0 | # Bio-Protocol Content
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A SYBR Green-based Real Time RT-PCR Assay for Detection of the Emerging H7N9 Virus
ZZ Zheng Zhu
Lunbiao Cui
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1150 Views: 9277
Reviewed by: Vinay PanwarLee-Hwa Tai
Original Research Article:
The authors used this protocol in Nov 2013
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Abstract
Most recently a novel avian-origin influenza A (H7N9) virus emerged in China and has been associated with lots of human infection and fatal cases. Molecular diagnostic methods are thus urgently needed in public health laboratories. We developed a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR) to detect the novel H7N9 virus.
Materials and Reagents
Respiratory specimens (throat-swabs, sputum and tracheal aspirate) from patients with influenza-like illnesses
Madin-Darby canine kidney (MDCK) cells (Shanghai Institutes for Biological Sciences)
Eagle's Minimum Essential Medium (EMEM) (Life Technologies, Gibco®, catalog number: 11095-080 )
Fetal bovine serum (Life Technologies, Gibco®, catalog number: 10099 )
Viral transportation medium (Yocon Bio-technology, catalog number: MT0301-1 )
QIAsymphony Virus/Bacteria Mini Kit (QIAGEN, catalog number: 931036 )
Nuclease-free water (Life Technologies, catalog number: 10977 )
SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies, catalog number: 11736 )
The primer sequences used for detection of influenza A H7N9 virus
Primera
Sequence (5’-3’)b
PCR amplicon (bp)
H7F
H7R
N9F
N9R
TGAAAATGGVTGGGAAGGYY
TGCCGATTGRGTGCTYTTRT
ACAGTGTACAAYAGCARRGT
GTTTCGRGCCCAYGTRTTAA
103
165
a F: Forward; R: Reverse
b V: A/C/G; Y: C/T; R: A/G
Equipment
T-25 cell culture flask (Corning, catalog number: 430168 )
A biosafety 3 laboratory (BSL-3) (required for virus isolation)
QIAsymphony SP instrument (QIAGEN, catalog number: 9001297 )
ABI Prism 7900HT Sequence Detection System (Life Technologies, Applied Biosystems®, catalog number: 4329001 )
Software
Sequence Detection System Software (v2.3) (Life Technologies, Applied Biosystems®)
Procedure
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Category
Microbiology > Microbial genetics > RNA
Molecular Biology > RNA > qRT-PCR
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1,151 | https://bio-protocol.org/exchange/protocoldetail?id=1151&type=0 | # Bio-Protocol Content
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EML Erythroid and Neutrophil Differentiation Protocols
Cristina Pina
CF Cristina Fugazza
TE Tariq Enver
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1151 Views: 11445
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Mar 2012
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Abstract
Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML cell line is conferred by the presence of a dominant-negative retinoic acid receptor (RAR) originally delivered by retroviral transduction (Tsai et al., 1994), which arrests cells at an early progenitor stage blocked from normal progression into myeloid differentiation. The presence of the RAR trans-gene does not interfere with erythroid differentiation, and it is possible to capture a low percentage of early erythroid, but not myeloid, committed cells in maintenance cultures (Pina et al., 2012; Ye et al., 2005).
Cells can be driven into granulocytic/neutrophil differentiation through the use of high doses of retinoic acid (RA), which overcomes the differentiation block. It should be noted that these pharmacological doses of RA are not compatible with erythroid differentiation, and it is hence not viable to obtain robust erythroid and myeloid differentiation in the same assay. Indeed, colonies scored as mixed-lineage in CFC assays are a mixture of undifferentiated and erythroid cells (Tsai et al., 1994). Nevertheless, robust single-lineage erythroid and neutrophil differentiation can be obtained in liquid culture under defined cytokine conditions, as specified below.
Materials and Reagents
IMDM (powder or liquid)
Horse serum (HS) (may require batch testing if low cell viability)
Foetal bovine serum (FBS)
L-Glutamine
Penicillin/Streptomycin (P/S)
SCF-conditioned medium (SCF-CM) (see Notes)
Recombinant human erythropoietin (e.g. epoietin alpha, Amgen) (obtained through hospital pharmacy under appropriate local guidelines)
Recombinant mouse interleukin-3 (IL-3) (e.g. Pepro Tech, catalog number: 213-13 )
Recombinant mouse granulocyte-monocyte colony-stimulating factor (GM-CSF) (e.g. Pepro Tech, catalog number: 315-03 )
All-Trans Retinoic Acid (ATRA) (e.g. Sigma-Aldrich, catalog number: 302-79-4 , reconstitute in ethanol)
Culture supplements and antibiotics (L-Glutamine and Penicillin/Streptomycin)
Monoclonal anti-mouse antibodies for flow cytometry
C-kit/CD117 (clone 2B8) (e.g. PE-Cy7, eBioscience, catalog number: 25-1171 ) (suggested use at 1:100 dilution)
CD34 (clone RAM34) (e.g. Alexa-Fluor 647, eBioscience, catalog number: 51-0341 ) (suggested use at 1:100 dilution)
Mac-1/CD11b (clone M1/70) (e.g. PE, eBioscience, catalog number: 12-0112 ) (suggested use at 1:100 dilution)
Gr1/Ly6C (clone RB6-8C5) (e.g. FITC, eBioscience, catalog number: 11-5931 ) (suggested use at 1:100 dilution)
Sca-1/Ly6A/E (clone D7) (e.g. Pacific blue, BioLegend, catalog number: 122520 ) (suggested use at 1:50 dilution)
Note: Stain on ice, in culture medium, for 20 min; wash with 10-20x volume of medium; pellet cells at 400 x g for 5 min. Re-suspend in 300-500 μl of medium for FACS analysis.
Trypan blue
Equipment
T175 tissue culture flasks with filtered cap (for production of conditioned medium)
T75 or T25 tissue culture flasks with filtered cap, or 6-well plates
Note: For EML cultures, the size of the tissue culture vial is determined by the cell number seeded at the cell densities indicated in the protocol.
Cell culture incubator
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pina, C., Fugazza, C. and Enver, T. (2014). EML Erythroid and Neutrophil Differentiation Protocols. Bio-protocol 4(12): e1151. DOI: 10.21769/BioProtoc.1151.
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Category
Cell Biology > Cell isolation and culture > Cell differentiation
Stem Cell > Adult stem cell > Hematopoietic stem cell
Stem Cell > Adult stem cell > Maintenance and differentiation
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1,152 | https://bio-protocol.org/exchange/protocoldetail?id=1152&type=0 | # Bio-Protocol Content
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MTV1 Pull-down Assay in Arabidopsis
Michael Sauer
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1152 Views: 12154
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Jun 2013
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Abstract
This protocol is an example of how to analyse suspected interactions between proteins using a pull-down assay (Sauer et al., 2013). A bait protein of interest (in this case, MTV1 of Arabidopsis thaliana) is fused to a GST tag and expressed in bacteria. The protein is isolated and allowed to bind to a matrix of glutathione-conjugated agarose beads via the GST-tag. Unspecifically binding proteins from the bacterial lysate are removed from the matrix. A native plant protein extract is then passed over the matrix and binding between the bait GST-MTV1 and prey proteins can occur. Extensive washes remove unspecifically bound proteins and finally, bait and prey proteins are released from the beads. Immunoblot analysis is then used to identify the proteins that bound to GST-MTV1. Importantly, a negative control consisting of the GST-tag alone is analysed in parallel to exclude the possibility that prey protein binding to the GST-MTV1 bait was due to the GST-tag.
Keywords: Pull-down Interaction Clathrin MTV1 EPSIN
Materials and Reagents
BL21 Escherichia coli (E. coli) cells containing a plasmid for expression of the recombinant GST-MTV1 fusion protein (the bait)
Note: In this exemplary case, the MTV1 coding sequence was cloned into a modified pGEX-2T plasmid (General Electric Company, catalog number: 28-9546-53 ), in which the multiple cloning site had been replaced by a Gateway cloning cassette (Note 1).
BL21 E. coli cells expressing the GST tag alone as negative control
Murashige and Skoog medium mix with vitamins and MES buffer (Duchefa Biochemie BV, catalog number: M0255.0010 )
Glutathione agarose (Sigma-Aldrich, catalog number: G4510 )
Complete inhibitor (EDTA free) (Roche Diagnostics, catalog number: 11 873 580 001 )
Phenylmethylsulfonyl fluoride PMSF (e.g. Sigma-Aldrich, catalog number: P7626 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Carbenicillin (e.g. Sigma-Aldrich, catalog number: C9231 ) (Note 2)
Isopropyl β-D-1-thiogalactopyranoside (IPTG) (e.g. Sigma-Aldrich, catalog number: I6758 )
Note: Should be prepared as aqueous stock solution of 1 M and stored at -20 °C.
Sodium dodecyl sulfate (SDS) (e.g. Sigma-Aldrich, catalog number: L3771 , or any other supplier)
Note: An aqueous stock solution of 20% (weight/volume) can be prepared and sterilized by autoclaving at 121 °C for 15 min.
Glycerol (e.g. Sigma-Aldrich, catalog number: G5516 , or any other supplier)
Tris (hydroxymethyl) aminomethane (Tris) (e.g. Sigma-Aldrich, catalog number: 252859 , or any other supplier)
Yeast extract (e.g. Sigma-Aldrich, catalog number: Y1625 , or any other supplier)
Tryptone (e.g. Sigma-Aldrich, catalog number: 95039 , or any other supplier)
Anti-GST polyclonal antibody (optional) (Carl Roth, catalog number: 3998 )
Anti-CHC monoclonal antibody (optional) (BD Biosciences, catalog number: 610499 )
Liquid nitrogen
NaCl (any supplier)
KCl (any supplier)
Na2HPO4 (any supplier)
KH2PO4 (any supplier)
β-mercaptoethanol
Liquid grown Arabidopsis seedlings of 6-8 days (see Recipes)
MS medium (see Recipes)
PBS (see Recipes)
Wash buffer (see Recipes)
Extraction buffer (see Recipes)
Sample loading buffer (see Recipes)
Liquid Lysogeny Broth (LB) growth medium (see Recipes)
Equipment
Microcentrifuge for 1.5 and 2 ml standard reaction tubes (any manufacturer)
Note: Either refrigerated or situated in a 4 °C cold-room, should be able to reach 16,000 x g.
Refrigerated centrifuge for 50 ml conical “Falcon” type tubes (any manufacturer)
Note: Should reach 4 °C and 3,000 x g.
Sonicator device (micro tip sonotrode type)
Note: We use the labsonic model of B. Braun, which is, however, no longer produced. But any tip style sonicator device that is suitable for small volumes (2-5 ml) will work, for example the UP100H device coupled to the MS3 sonotrode (Hielscher Ultrasound Technology).
Poly-Prep Chromatography columns (Bio-Rad Laboratories, catalog number: 731-1550 )
Erlenmeyer flasks
Paper towels
1.5 ml microcentrifuge tubes capable of supporting 16,000 x g (any supplier)
50 ml polypropylene conical centrifuge tubes “Falcon” type (any supplier)
0.20 µm filter unit (e.g. Minisart®, Sartorious, catalog number: 17597 ) plus compatible 5 ml syringe
Shaking incubator for bacteria 37 °C (any manufacturer)
Shaking incubator 25 °C or orbital shaker at room temperature (any manufacturer)
End-over-end (orbital) mixer (any manufacturer)
Spectrophotometer capable of measuring optical density at 600 nm (any manufacturer)
Mortar and pestle (about 10 cm diameter) (any manufacturer)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sauer, M. (2014). MTV1 Pull-down Assay in Arabidopsis. Bio-protocol 4(12): e1152. DOI: 10.21769/BioProtoc.1152.
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Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Expression
Biochemistry > Protein > Interaction
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1,153 | https://bio-protocol.org/exchange/protocoldetail?id=1153&type=0 | # Bio-Protocol Content
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Determination of Oxygen Respiration Rates in Wetted Developmentally Arrested Spores of Streptomyces Species
Marco Fischer
Dörte Falke
R. Gary Sawers
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1153 Views: 8298
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
Streptomyces species produce spores, which, while not as robust as endospores of Bacillus or Clostridium species, are capable of surviving for months or even years (Hopwood, 2006). During this time these spores remain viable, surviving by slowly degrading internal stores of carbon compounds, such as the carbohydrate trehalose. To enable metabolism to continue they must have access to an electron acceptor that allows the removal of the reducing equivalents that accumulate through metabolic activity. The most commonly used acceptor is oxygen. We describe the quantitative measurement of oxygen respiration rates by developmentally arrested spores of the streptomycete Streptomyces coelicolor (Fischer et al., 2013).
Keywords: Cytochrome oxidase Oxygen reduction Spores Non-invasive optical oxygen-sensing Streptomyces coelicolor
Materials and Reagents
Freshly harvested Streptomyces spores in water
Soya flour (from local supermarket)
D-mannitol (Sigma-Aldrich, catalog number: 63560 )
Agar-agar (Kobe I) (Roth North America)
3-(N-morpholino) propanesulfonic acid (MOPS) buffer (Roth North America)
Chloramphenicol (Merck KGaA)
Sodium hydroxide pellets (Roth North America)
MOPS-buffer (see Recipes)
SFM agar (see Recipes)
Equipment
Cotton wool (standard issue from local Pharmacy) for spore preparation
0.22 µm pore-size filters (PVDF) (Sartorius AG)
30 °C rotary shaker
Baffled Erlenmeyer flasks (500 ml) (Glasgerätebau Ochs, Laborfachhandel e. K., catalog number: 100500 )
Standard-sized plastic Petri dishes for bacterial growth and spore preparation
Gas-tight glass Hungate tubes (16 ml) for anaerobic work with butyl rubber septa (Glasgerätebau Ochs, Laborfachhandel e. K., catalog number: 1020471 )
Cooled table-top centrifuge (e.g. Eppendorf)
Needles (0.6 mm gauge) (B. Braun Melsungen AG)
Water bath or heating block (Biometra)
Spectrophotometer capable of measuring absorption in the visible range
Optical ‘FirestingO2 oxygen meter’ (Pyro Science GmbH)
Contactless optical oxygen ‘sensor spot’ (Pyro Science GmbH)
‘Bare fibre’ (Pyro Science GmbH)
Adjustable mini magnetic stirrer (e.g. IKA) (10 mm x 0.3 mm magnetic stirrer bar) (e.g. Roth North America)
Software
Firesting Logger Software (Pyro Science GmbH)
Procedure
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How to cite：Fischer, M., Falke, D. and Sawers, R. G. (2014). Determination of Oxygen Respiration Rates in Wetted Developmentally Arrested Spores of Streptomyces Species. Bio-protocol 4(12): e1153. DOI: 10.21769/BioProtoc.1153.
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Category
Microbiology > Microbial biochemistry > Other compound
Microbiology > Microbial metabolism > Nutrient transport
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1,154 | https://bio-protocol.org/exchange/protocoldetail?id=1154&type=0 | # Bio-Protocol Content
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Rapid Nitrate Reduction Assay with Intact Microbial Cells or Spores
Marco Fischer
Dörte Falke
R. Gary Sawers
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1154 Views: 8200
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
Many microorganisms have the capacity to use nitrate as a respiratory electron acceptor. Reduction of nitrate is catalyzed by a multi-subunit nitrate reductase that is often located associated with the cytoplasmic membrane and has its active site oriented toward the cytoplasm. This means that nitrate must be transported into the cell and often this occurs concomitantly with the export of the reduced nitrite product. Often nitrate and nitrite transport are coupled through the action of a nitrate: nitrite antiporter. Microbial cells, spores and mycelium harbour intracellular storage compounds such as trehalose or glycogen that, upon metabolism, function as endogenous electron donors for nitrate reduction. It is also possible to use glucose supplied exogenously as a substrate for nitrate reduction. The method described here allows the direct analysis of nitrate reduction by whole cell material without the requirement for artificial electron donors. This method is also applicable to the study of spores, particularly those of Streptomyces species (Fischer et al., 2013). The paper by Fischer et al. 2013 provides examples of datasets for the method presented below.
Keywords: Nitrate reductase Metabolism Nitrite Respiration Dormancy
Materials and Reagents
Freshly harvested Streptomyces spores in water or mycelium in 50 mM MOPS buffer (pH 7)
Tryptic Soy Broth (TSB) (Sigma-Aldrich, catalog number: 22092 )
3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Roth North America)
Sulfanilic acid (Sigma-Aldrich, catalog number: 251917 )
N-(1-naphthyl)-ethylenediamine dihydrochloride (Roth North America)
Hydrochloric acid (Roth North America)
Sodium hydroxide pellets (Roth North America)
Soya flour (from local supermarket)
D-mannitol (Sigma-Aldrich catalogue number 63560 )
Agar-agar (Kobe I) (Roth North America)
Sulfanilic acid solution (see Recipes)
N-(1-naphthyl)-ethylenediamine solution (see Recipes)
MOPS-buffer (see Recipes)
SFM agar (see Recipes)
Equipment
Cotton wool (standard issue from local pharmacy)
0.22 µm pore-size filters (PVDF) (Roth North America)
30 °C rotary shaker
Baffled Erlenmeyer flasks (500 ml) (Glasgerätebau Ochs, Laborfachhandel e. K., catalog number: 100500 )
Standard-sized plastic Petri dishes for bacterial growth and spore preparation
Gas-tight glass Hungate tubes (16 ml) for anaerobic work with butyl rubber septa (Glasgerätebau Ochs, Laborfachhandel e. K., catalog number: 1020471 )
Cooled table-top centrifuge (e.g. Eppendorf)
Cylinder of pure nitrogen gas (e.g. Linde or local supplier)
Needles 0.6 mm Gauge (B. Braun Melsungen AG)
Water bath or heating block (Biometra)
Spectrophotometer measuring absorption in the visible range
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Fischer, M., Falke, D. and Sawers, R. G. (2014). Rapid Nitrate Reduction Assay with Intact Microbial Cells or Spores. Bio-protocol 4(12): e1154. DOI: 10.21769/BioProtoc.1154.
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Category
Microbiology > Microbial biochemistry > Other compound
Microbiology > Microbial metabolism > Nutrient transport
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1,155 | https://bio-protocol.org/exchange/protocoldetail?id=1155&type=0 | # Bio-Protocol Content
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Deflagellation and Regeneration in Chlamydomonas
WD William Dentler
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1155 Views: 14325
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Jan 2013
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Abstract
Eukaryotic cilia/flagella are one of the only cellular structures that can be removed without injuring cells, can be highly purified for biochemical analysis, and, in many cells, can be completely reassembled within 90 minutes. Following amputation, the expression of many flagellar genes is up-regulated, and many are packaged and associated with intraflagellar transport (IFT) particles for transport to flagellar bases and into growing flagella. Studies of deciliation and ciliary growth provide insight to mechanisms that regulate microtubule assembly and length, mechanisms that regulate the transport of soluble cytoplasmic proteins into the ciliary compartment and their assembly into microtubules, and mechanisms that regulate trafficking of membrane proteins and lipids to the plasma membrane or to ciliary bases and their movement into and out of the cilium. These are important for motility and for signal transduction.
Deciliation methods for many cells have been developed and most require extracellular calcium ions and activation of signaling pathways that regulate microtubule severing (Quarmby, 2009). Deciliation occurs at the distal end of the basal bodies and, as soon as axonemes are severed, the membrane reseals and basal bodies begin to regenerate cilia.
Chlamydomonas is an ideal organism with which to study ciliary regeneration. Cells are easily and inexpensively cultured, flagellar amputation and regeneration is uniform in all cells in a population and growth can be assayed by observing fixed or living cells with a phase contrast microscope equipped and a 40x objective lens. Flagellar regeneration on individual living cells can be observed using paralyzed mutants immobilized in agarose. Because deflagellation leaves cells intact, the released flagella can be purified without contamination with cellular debris. The most reliable deciliation and regeneration method is the pH shock method developed by Reference 5 (also see References 4 and 11). Other methods are reviewed by Quarmby, (2009). The pH shock method is primarily used for Chlamydomonas but can be used for deciliation and regeneration of Tetrahymena cilia (Gaertig et al., 2013).
Figure 1. Typical flagellar regeneration curve showing phase contrast images of Chlamydomonas cells photographed before deflagellation and during regeneration. The average flagellar lengths of a population of regenerating cells is shown in red and the average flagellar lengths on a population of nondeflagellated cells is in blue.
Materials and Reagents
Cells
Chlamydomonas cells can be obtained from a variety of sources and pure strains of a variety of flagellar mutants can be obtained from the Chlamydomonas Resource Center (http://chlamycollection.org/contact-us/).
Media and cell culture (see Notes)
0.5 N acetic acid
0.5 M NaOH
2% glutaraldehyde in M medium or Lugols iodine (see Recipes)
Note: For electron microscopy, one should use fresh glutaraldehyde from sealed ampuoles. To measure flagellar lengths, the age of the glutaraldehyde is not critical.
Optional:
2.5% low EEO agar (Thermo Fisher Scientific, FisherBiotechTM, catalog number: BP160-100 )
Note: It is used to observe flagellar growth or maintenance on individual living cells.
5 mM (final concentration) colchicine (to inhibit microtubule assembly)
Note: These experiments are carried out in phosphate-buffered Minimal medium. Avoid Tris-containing buffers because they may inhibit the effects of colchicine (Margulis et al., 1969).
10 µg/ml (final concentration) Cycloheximide
Note: It is used to inhibit protein synthesis.
VALAP (see Recipes)
Note: It is used to support coverslips to examine living cells without inducing deflagellation by coverslip pressure.
Equipment
pH meter calibrated for pH 4-7
Note: Some gel-filled electrodes are not accurate across this pH range.
Centrifuge and tubes
Note: For small scales, a clinical centrifuge and conical tubes.
Phase contrast microscope or brightfield microscope
Note: If cells are fixed with Lugol’s iodine, a 40x lens is ideal for flagellar length measurements.
Orbital shaker
Magnetic stirrer
Stir bar
Software
Image J (http://imagej.nih.gov/ij/)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Dentler, W. (2014). Deflagellation and Regeneration in Chlamydomonas. Bio-protocol 4(12): e1155. DOI: 10.21769/BioProtoc.1155.
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Category
Plant Science > Phycology > Cell analysis
Cell Biology > Cell movement > Cell motility
Cell Biology > Cell imaging > Live-cell imaging
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1,156 | https://bio-protocol.org/exchange/protocoldetail?id=1156&type=0 | # Bio-Protocol Content
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Ciliary and Flagellar Membrane Vesicle (Ectosome) Purification
WD William Dentler
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1156 Views: 9633
Edited by: Ru Zhang
Reviewed by: Lin FangFanglian He
Original Research Article:
The authors used this protocol in Jan 2013
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Abstract
Eukaryotic cilia/flagella are ideal organelles for the analysis of membrane trafficking, membrane assembly, and the functions of a variety of signal transduction molecules. Cilia are peninsular organelles and the membrane lipids, membrane proteins, and microtubular-associated components are selectively transported into cilia through the region formed by the basal body/transition region and tightly associated ciliary membrane. Cilia can be isolated from many organisms without disrupting cells and many will rapidly regenerate cilia (with the ciliary membrane lipids and proteins) to replace those that are released. Despite their ease of isolation, we have relatively little understanding of the mechanisms that regulate lipid and protein transport into ciliary membranes (Pazour and Bloodgood, 2008; Bloodgood, 2009; Bloodgood, 2012).
Chlamydomonas flagella shed membrane vesicles, also called ectosomes (Wood et al., 2013) from flagellar tips and these vesicles can be purified from the culture medium without damaging or deflagellating cells (McLean et al., 1974; Bergman et al., 1975; Snell, 1976; Kalshoven et al., 1990). Based on a comparison of biotinylated proteins on the shed vesicles with biotinylated proteins isolated from purified flagella and cell bodies, the ectosomes contain most, but not all, flagellar surface proteins and none of the major cell body proteins (Dentler, 2013). Although ectosomes have only been purified from Chlamydomonas cells, preliminary evidence indicates that similar vesicles are released from Tetrahymena cilia (Dentler, unpublished).
Flagellar (and ciliary) membranes or membrane proteins also can be released from purified flagella/cilia. Most membrane proteins can be solubilized by extracting purified cilia with nonionic detergent [Triton X-100 or X-114 or Nonidet P-40 (NP-40)] and pelleting the microtubules (axonemes). However, not all membranes are released by detergent (Dentler, 1980) and the supernatant also contains all of the flagellar proteins that are not attached to the microtubules.
Intact membrane vesicles can be released from flagella by agitation of flagella, often with low concentrations of nonionic detergents or freeze-thawing (Witman et al., 1972; Snell, 1976; Dentler, 1980; Dentler, 1995; Bloodgood and May, 1982; Pasquale and Goodenough, 1987; Iomini et al., 2006; Huang et al., 2007). Once released, they can be purified from axonemes by differential centrifugation.
Each of these methods may enrich for different populations of axonemal and membrane proteins and lipids. The different solubility of membranes may reveal local differences in lipid or protein composition (Bloodgood, 2009). The ectosomes contain most but not all surface proteins found on purified Chlamydomonas flagella (Dentler, 2013). The ectosomes vesicles may be enriched in different soluble flagellar proteins than those trapped as vesicles are released from purified flagella. The detergent-solubilized “membrane+matrix” will contain all soluble membrane proteins as well as all of the soluble proteins in the flagellar compartment.
In this paper, a method to purify ectosomes vesicles released from the tips of living Chlamydomonas cells is presented as are two methods to release flagellar membrane vesicles and proteins from purified flagella.
Figure 1. A: Purified flagella (phase contrast); B: Purified flagella (TEM); C: Shed membrane vesicles (TEM). Bars = 500 nm
Materials and Reagents
Cells
Chlamydomonas cells can be obtained from a variety of sources and pure strains can be obtained from the Chlamydomonas Resource Center (http://chlamycollection.org/contact-us/).
Media and cell culture (see Notes)
Culture media for washing cells (2-4 L)
0.5 N acetic acid
0.5 N KOH
Sucrose
Triton X-100 or Nonidet P-40
Cilia wash buffer (CWB) (see Recipes)
Equipment
Pellicon tangential flow microfiltration cassettes (Millipore, www.millipore.com)
Note: Cells can be harvested using 450 ml centrifuge bottles and large rotor but, for 8-16 L of cells, harvesting is more rapid and fewer cells are deflagellated using a Pelicon.
Preparative centrifuge with rotors for 450 ml bottles and for 40 ml tubes
450 ml centrifuge bottles
40 ml centrifuge tubes
12 ml centrifuge tubes
Medium speed centrifuge with swinging bucket rotor and angle rotor
Fernbach flasks (2,800 ml) or large flasks
Ultracentrifuges and rotors
Note: For 750 ml, I use Beckman 35 or Ty45Ti rotors and two Beckman ultracentrifuges.
Phase contrast microscope
Orbital shaker
Bright fluorescent light - generally 4-6 F20/40 PL/AQ lamps
Transmission electron microscope
Magnetic stirrer
pH electrode
Procedure
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Category
Plant Science > Phycology > Cell analysis
Cell Biology > Cell movement > Cell motility
Cell Biology > Organelle isolation > Membrane
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1,157 | https://bio-protocol.org/exchange/protocoldetail?id=1157&type=0 | # Bio-Protocol Content
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In organello Protein Synthesis
Malgorzata Kwasniak-Owczarek
Hanna Janska
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1157 Views: 12814
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in May 2013
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Abstract
In organello protein synthesis method allows the analysis of mitochondrial translation products. The principle of this method relies on incubation of isolated intact mitochondria with radiolabeled amino acids such as 35S methionine. After protein synthesis, the radiolabeled translation products are subsequently separated by SDS polyacrylamide gel electrophoresis and analysed by autoradiography. For in organello analysis of protein synthesis, the isolated intact mitochondria must retain their bioenergetics capacity, and in consequence be fully functional and able to perform coupled respiration. This in turn requires a quick and gentle purification of mitochondria during their isolation.
Keywords: In organello method Protein synthesis Mitochondria Arabidopsis thaliana
Materials and Reagents
Youngest leaves harvested from 9- to 10-week-old Arabidopsis thaliana
Common chemicals
Klorin, NaCl, Tween-20, sucrose, tetrasodiumpyrophosphate, PVP-40, EDTA, KH2PO4, sodium ascorbate, L-cysteine, TES, BSA, GTP, mannitol, KCl, DTT, HEPES, MgCl2, sodium acetate, ADP, malic acid, pyruvate, puromycin, L-methionine, isopropanol, acetic acid, Commassie Blue G-250, glycerol, SDS, Tris, β-mercaptoethanol, bromophenol blue
Sand (50-70 mesh particle size) (Sigma-Aldrich, catalog number: 274739 )
Percoll (pH 8.5-9.5) (25 °C) (Sigma-Aldrich, catalog number: P1644 )
L-35S methionine (HARTMANN ANALYTIC, catalog number: SRM-01H )
Amino acid mixture without L-methionine (Promega Corporation, catalog number: L9961 )
DC Protein Assay Kit (Bio-Rad Laboratories, catalog number: 500-0112 )
12% SDS-polyacrylamide gel (Leammli electrophoresis system)
10% chlorox solution (see Recipes)
Grinding medium (see Recipes)
2x wash buffer (see Recipes)
Synthesis mix (see Recipes)
Stop solution (see Recipes)
Isopropanol fixing solution (see Recipes)
Rapid Coomassie Blue G-250 staining solution (see Recipes)
Destaining solution (see Recipes)
1x solubilzation buffer (see Recipes)
Equipment
Mortar
Miracloth (Calbiochem®, catalog number: 475855-1R )
Polycarbonate centrifuge tubes with round bottom (30 ml and 90 ml)
1.5 ml microcentrifuge tubes (SARSTEDT AG, catalog number: 72.690.001 )
Paint brushes
Tubes with round bottom (SARSTEDT AG, catalog number: 55.484.001 )
Pasteur pipette
Gradient former model 485 (Bio-Rad Laboratories, catalog number: 165-4120 )
Peristaltic pump –PumpP-1 (GE Healthcare, catalog number: 18-111--91 )
Centrifuges 1K15 and 3K18 (Sigma-Aldrich)
Microcentrifuges (Eppendorf, catalog number: 5452 000.018 )
Incubator shaker (IKA KS4000i control shaker, catalog number: 3510001 )
Spectrophotometer UV-1800 (Schimadzu, catalog number: 206-25400-32 )
SDS-PAGE system - Mini-Protean Tetra Cell (Bio-Rad Laboratories, catalog number: 165-8000 )
Slab Gel dryer SGD5040 (Thermo Fisher Scientific, catalog number: SGD5040-230 )
Radioactive room
Carestream Kodak BioMax MR film (Sigma-Aldrich, catalog number: Z353949-50EA )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kwasniak-Owczarek, M. and Janska, H. (2014). In organello Protein Synthesis. Bio-protocol 4(12): e1157. DOI: 10.21769/BioProtoc.1157.
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Category
Plant Science > Plant biochemistry > Protein
Cell Biology > Organelle isolation > Mitochondria
Molecular Biology > Protein > Detection
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1,158 | https://bio-protocol.org/exchange/protocoldetail?id=1158&type=0 | # Bio-Protocol Content
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Peer-reviewed
ChIP-Seq in Candida albicans
Sadri Znaidi
CP Caroline Proux
SW Sandra Weber
SD Simon Drouin
FR François Robert
MR Martine Raymond
JC Jean-Yves Coppée
Cd Christophe d’Enfert
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1158 Views: 17730
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
Systems biology approaches can be used to study the regulatory interactions occurring between many components of the biological system at the whole-genome level and decipher the circuitries implicated in the regulation of cellular processes, including those imparting virulence to opportunistic fungi. Candida albicans (C. albicans) is a leading human fungal pathogen. It undergoes morphological switching between a budding yeast form and an elongated multicellular hyphal form. This transition is required for C. albicans’ ability to cause disease and is regulated through highly interconnected regulatory interactions between transcription factors (TFs) and target genes. The chromatin immunoprecipitation (ChIP)-High-throughput sequencing (Seq) technology (ChIP-Seq) is a powerful approach for decoding transcriptional regulatory networks. This protocol was optimized for the preparation of ChIP DNA from filamenting C. albicans cells followed by high-throughput sequencing to identify the targets of TFs that regulate the yeast-to-hyphae transition.
Keywords: Candida albicans Chromatin immunoprecipitation Transcriptional regulatory networks Transcription factors Systems biology
Materials and Reagents
C. albicans strains expressing or not a functional epitope-tagged transcription factor grown under filamentation-inducing conditions in liquid medium [50 ml, e.g. Lee’s medium at 37 °C (Lee et al., 1075)]
37% formaldehyde (Sigma-Aldrich, catalog number: F8775 )
Liquid nitrogen
Dynabeads® Pan Mouse IgG (5 ml) (Life Technologies, catalog number: 11041 )
Appropriate mouse monoclonal antibody directed against the epitope tag fused to TF [e.g. mouse monoclonal anti-HA antibody, HA-probe Antibody (F-7), Santa Cruz, catalog number: sc-7392 ]
100 mM Phenylmethylsulfonyl fluoride (PMSF) dissolved in isopropanol (100x stock solution)
DNase-free RNase A solution (10 mg/ml) (Thermo Fisher Scientific, catalog number: EN0531 )
Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: B4287 )
Proteinase K solution (20 mg/ml) (Life Technologies, catalog number: AM2546 )
Glycogen (20 mg/ml) (Thermo Fisher Scientific, catalog number: FERR0561 )
Phenol: Chloroform: Isoamyl alcohol (25: 24: 1) (Sigma-Aldrich, catalog number: P2069 )
5 M NaCl solution
70%, 100% freezer-cold Ethanol
Quant-iTTM PicoGreen® dsDNA Assay Kit (Life Technologies, catalog number: P11496 )
TruSeqTM DNA Sample Preparation Kit v.2 (Illumina, catalog numbers: FC-121-2001 , FC-121-2002 )
TruSeqTM DNA Sample Preparation Guide v.2 (Illumina, catalog number: FC-930-1021)
QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )
MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
E-Gel® iBaseTM and E-Gel® Safe ImagerTM Combo Kit (Life Technologies, catalog number: G6465 )
2% E-Gel® SizeSelectTM Agarose Gels (Life Technologies, catalog number: G6610-02 )
Glycine (Sigma-Aldrich, catalog number: 50046 )
SDS (Sigma-aldrich, catalog number: L3771 )
2.5 M glycine (see Recipes)
10% SDS (see Recipes)
Lee’s medium (see Recipes)
TBS buffer (see Recipes)
Lysis buffer (see Recipes)
PBS/0.5% BSA (see Recipes)
Wash buffer (see Recipes)
Protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11697498001 ) (see Recipes)
TE solution (see Recipes)
TE/SDS solution (see Recipes)
Equipment
15 ml, 50 ml Falcon tubes
Racks, including a 50-ml Falcon tube rack (Unwire test tube rack, for 30 mm tubes; holds 24) (Thermo Fisher Scientific, Nalgene®, catalog number: 14-809-30 )
VSR-50 laboratory platform rocker (Pro Scientific, catalog number: PSI-512000-00 ) or equivalent
Waste container
1.5 ml screw-cap tubes
0.5 ml PCR tube
FastPrep®-24 instrument (MP Biomedicals, catalog number: 116004500 )
Microscope (e.g. Zeiss Axiostar Plus)
18 G x 1½ inch needles (BD Biosciences, catalog number: 305196 )
2 ml Eppendorf conical tubes (Eppendorf, catalog number: 022363352 )
Probe sonicator (e.g. MSE Soniprep 150 Plus, exponential microprobe, end diameter 3 mm) [MSE (UK), catalog number: 38121-114A ]
Glass beads (0.5 mm diameter) (Bio Spec Products, catalog number: 11079105 )
Hematology/chemistry mixer 346 (Thermo Fisher Scientific, catalog number: 14-059-346 )
DynaMag Spin magnet system (Life Technologies, model: 123-20D )
Fluorescence reader (e.g. Tecan Trading AG, Infinite®, model: M200 )
BioAnalyzer 2100 (Agilent)
HiSeq 2000 sequencer (Illumina)
Software
Galaxy NGS data analysis software (https://main.g2.bx.psu.edu/)
ChIP-Seq (MACS) peak-finding algorithm software (http://liulab.dfci.harvard.edu/MACS/)
Procedure
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Copyright: © 2014 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:
Znaidi, S., Proux, C., Weber, S., Drouin, S., Robert, F., Raymond, M., Coppée, J. and d’Enfert, C. (2014). ChIP-Seq in Candida albicans. Bio-protocol 4(12): e1158. DOI: 10.21769/BioProtoc.1158.
Znaidi, S., Nesseir, A., Chauvel, M., Rossignol, T. and d'Enfert, C. (2013). A comprehensive functional portrait of two heat shock factor-type transcriptional regulators involved in Candida albicans morphogenesis and virulence. PLoS Pathog 9(8): e1003519.
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Category
Systems Biology > Genomics > ChIP-seq
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Immunodetection
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1,159 | https://bio-protocol.org/exchange/protocoldetail?id=1159&type=0 | # Bio-Protocol Content
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Analysis of Mycobacterial Protein Secretion
Alka Mehra
P Jennifer A. Philips
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1159 Views: 13081
Reviewed by: Fanglian HeRon Saar-Dover
Original Research Article:
The authors used this protocol in Oct 2013
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Abstract
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while others may be important antigens or serve as biomarkers of infection. Here, we describe a protocol to prepare whole cell extracts (WCE) and short term culture filtrate (CF) from Mtb or the vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) (Mehra et al., 2013). These are both slow growing mycobacteria, but the same basic procedure can easily be adapted to analyze secreted proteins from rapidly growing mycobacteria, such as Mycobacterium smegmatis (Msmeg), a non-pathogenic species commonly used in the laboratory. The fractions obtained can be analyzed by western blotting to examine proteins of interest or by mass spectrometry if antibodies are not available or to examine the entire secretome. Genetic knockout mutants for the gene of interest serve as a negative control. Additionally, levels of a cytosolic protein such as the chaperone GroEL or the pyruvate dehydrogenase E2 component sucB (Rv2215/dlaT) should be assessed in the CF fraction to rule out the possibility that a positive signal in CF is due to bacterial lysis (see Figure 1). By varying the growth conditions of the strain, this in vitro secretion assay can be used to examine conditions that alter the secretome. We are thankful to Magnus Stiegedal for helpful tips on TCA (trichloroacetic acid) precipitation.
Keywords: Mycobacteria culture Culture filtrates Whole cell extracts TCA precipitation Bead beating
Figure 1. Western analysis of secretion of EsxH by BCG. BCG containing an empty vector control and EsxG-EsxH-FLAG expression construct (FLAG tag at C terminal of EsxH) were analyzed for presence EsxH by anti-FLAG western in WCE and CF prepared as described in the protocol. DlaT was used as a loading control to indicate the degree of bacterial lysis.
Materials and Reagents
Note: All work with live Mtb must be performed in a Biosafety Level 3 (BSL3) facility.
Middlebrook 7H9 Broth (Difco, catalog number: 271310 )
Tween-80 (Sigma-Aldrich, catalog number: P4780 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Albumin-dextrose-catalase (ADC) (BD, catalog number: 212352 )
Oleic-albumin-dextrose-catalase (OADC) (BD, catalog number: 212351 )
Potassium phosphate (monobasic) (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
L-asparagine monohydrate (Sigma-Aldrich, catalog number: A8381 )
Citric acid monohydrate (Sigma-Aldrich catalog number: C1909 )
Ferric ammonium citrate (Sigma-Aldrich catalog number: F5879 )
Zinc sulfate monohydrate (ZnSO4.H2O) (Sigma-Aldrich, catalog number: 96495 )
Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: 230391 )
Chelex 100 resin (Bio-Rad Laboratories, catalog number: 142-2822 )
100x Halt Protease Inhibitor single use cocktail (Pierce, catalog number: 1860932 )
100% trichloroacetic acid (TCA) (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 49010 )
Acetone (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 32201 )
Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 10010-023 )
Bromophenol Blue, sodium salt (US Biological, catalog number: 12370 )
Tris (MP Biomedicals, catalog number: 02194855 )
Sodium Dodecyl Sulfate (SDS) (US Biological, catalog number: 18220 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
β-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M6250 )
7H9 complete media (see Recipes)
Sauton’s media (see Recipes)
Chelated sauton’s media (an alternative minimal media for mycobacterial growth) (see Recipes)
Protein extraction buffer (see Recipes)
5x SDS-PAGE sample buffer (see Recipes)
Equipment
Autoclave
Steriflip-GV filter units (0.22 µM pore size) (Millipore, catalog number: SE1M179M6 )
20 ml syringes (BD, catalog number: 302830 )
0. 22 µM syringe filter units (33 mm) (Millipore, catalog number: SLGV033RS )
Disposable Sterile Filter system (1L, 0.22 µm pore size) (Corning, catalog number: 09761104 )
0.1 mm zirconia/silica beads (Bio Spec Products, catalog number: 11079101z )
30 ml square media bottles (Nalgene®, catalog number: NE/2019-0030 )
125 ml square media bottles (Nalgene®, catalog number: NE/2019-0125 )
50 ml falcon tubes (Corning, catalog number 430290 )
15 ml falcon tubes (Corning, catalog number: 430052 ) with plug seal caps
Note: These 50 ml tubes are compatible with organic solvents and high speed centrifugation. Falcon tubes with these features can be used from different vendors.
Microtubes (2 ml screw cap with O rings) (SARSTEDT AG, catalog number: 72.693 )
Spectrophotometer
Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Beckman Coulter, model: Allegra X-15R ; bench top centrifuge with SX4750 rotor)
Notes:
Msmeg and BCG should be handled according to institutional standards of practice for biosafety.
Mtb cultures should be handled in biosafety level 3 facilities according to institutional standards of practice.
Centrifuging BCG and Mtb requires appropriate aerosol containment.
Beckman Aerosolve® canisters to contain aerosols during centrifugation of mycobacterial cultures (e.g. Beckman Coulter, catalog number: BK359232 )
37 °C shaking incubator
Aerosol containment units for shaking BCG and Mtb liquid cultures in the shaking incubator
Note: Incubator should be placed in BSL3 facility for Mtb cultures.
High speed centrifuge for 50 ml polypropylene falcons used for TCA precipitation of CF (e.g. Beckman Coulter centrifuge with JLA16.2 rotor)
50 ml falcon adaptors for rotor JLA16.2
Bead beater (Bio Spec Products, model: Minibead beater 16; http://www.biospec.com/product/34/mini_beadbeater/)
Standard table top centrifuge with refrigeration
Heating block for Eppendorf tubes (set to 95 °C)
pH meter
Acronyms
Mtb: Mycobacterium tuberculosis
BCG: Mycobacterium bovis bacillus Calmatte- Guérin
Msmeg: Mycobacterium smegmatis
WCE: Whole cell extract
CF: Culture filtrate
DlaT: Rv2215/pyruvate dehydrogenase E2 component sucB protein of mycobacteria
TCA: Trichloroacetic acid
BSC: Biosafety cabinet
OD600: Absorbance or Optical Density at wavelength of 600 nm
Procedure
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Copyright: © 2014 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:
Mehra, A. and Philips, J. A. (2014). Analysis of Mycobacterial Protein Secretion . Bio-protocol 4(12): e1159. DOI: 10.21769/BioProtoc.1159.
Mehra, A., Zahra, A., Thompson, V., Sirisaengtaksin, N., Wells, A., Porto, M., Koster, S., Penberthy, K., Kubota, Y., Dricot, A., Rogan, D., Vidal, M., Hill, D. E., Bean, A. J. and Philips, J. A. (2013). Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog 9(10): e1003734.
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Category
Microbiology > Microbial biochemistry > Protein
Microbiology > Microbial cell biology > Cell isolation and culture
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1,160 | https://bio-protocol.org/exchange/protocoldetail?id=1160&type=0 | # Bio-Protocol Content
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Determination of Luciferase Activity in Arabidopsis seedling
Mohan TC
GC Gabriel Castrillo
Antonio Leyva
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1160 Views: 12361
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
There are different direct and indirect methods available to study gene expression in plant systems. In this protocol we describe a modified expression assay using transgenic plants expressing the luciferase gene under the control of phosphate transporter PHT1;1 promoter. This assay was originally optimized for analyzing the repression of PHT1;1 promoter in response to arsenate As (V) which can be adapted to study the kinetics of transcriptional regulation of any gene in response to biotic or environmental stimuli measuring Luciferase activity in Arabidopsis thaliana.
Keywords: Luciferase Gene expression Arabidopsis
Materials and Reagents
Transgenic Arabidopsis seedlings expressing Luciferase fused to promoter of interest (i.e. PHT1;1:Luc in pGWB435 binary vector)
Note: Vector information can be find at: http://shimane-u.org/nakagawa/pgwb-tables/2.htm.
KH2PO4 (Sigma-Aldrich, catalog number: P5655 )
Sodium arsenate dibasic heptahydrate (Sigma-Aldrich, catalog number: A6756 )
30 mM arsenate (see Recipes)
0.36 mM D-Luciferin (Sigma-Aldrich, catalog number: L9504 ) (see Recipes)
Johnson media (see Recipes)
Equipment
Growth chamber [16-h-light/8-h-dark regime (24 °C/21 °C)]
White 96-well plates (Greiner Bio-One GmbH, catalog number: 655074 )
MicroAmp optical adhesive film (Life Technologies, Applied Biosystem®, catalog number: 4311971 )
Luminometer (centro LB 960) (Berthold Technologies)
Software
Mikro Win 2000 software
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：TC, M., Castrillo, G. and Leyva, A. (2014). Determination of Luciferase Activity in Arabidopsis seedling. Bio-protocol 4(12): e1160. DOI: 10.21769/BioProtoc.1160.
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Category
Plant Science > Plant molecular biology > DNA
Plant Science > Plant biochemistry > Protein
Molecular Biology > DNA > Gene expression
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1,161 | https://bio-protocol.org/exchange/protocoldetail?id=1161&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Bone Marrow Derived Eosinophil Cultures
TL Thomas X. Lu
Marc E. Rothenberg
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1161 Views: 13460
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Feb 2013
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Abstract
Eosinophils are multifunctional effector cells implicated in the pathogenesis of a variety of diseases including asthma, eosinophil gastrointestinal disorders and helminth infection. Mouse bone marrow derived progenitor cells can be differentiated into eosinophils following IL-5 exposure. These bone marrow derived eosinophils are fully differentiated at the end of a 14 day culture based on morphology and expression of molecular markers.
Materials and Reagents
Mice
Histopaque 1083 (Sigma-Aldrich, catalog number: 10831 -100ml)
IMDM with Glutamax-I (Life Technologies, InvitrogenTM, catalog number: 31980-097 )
Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
Penicillin/Streptomycin (Life Technologies, InvitrogenTM, catalog number: 15140-122 )
Stem Cell Factor (Pepro tech, catalog number: 250-03 )
FLT-3 Ligand (Pepro tech, catalog number: 250-31L )
Recombinant mouse IL-5 (Pepro tech, catalog number: 215-15 )
PBS (Life Technologies, InvitrogenTM, catalog number: 14200-166 )
Diff-Quick Stain Kit (Thermo Fisher Scientific, catalog numbers: 23-122-929 , 23-122-952 , and 23-122-937 )
RBC lysis buffer (Sigma-Aldrich, catalog number: R7757 -100ml)
CCR3 (R&D systems, Catalog number: FAB729F )
Siglec-F (BD, PharmingenTM, Catalog number: 552126 )
IMDM cell culture media (see Recipes)
Equipment
6-well tissue culture plate
Dissection tools: scalpel, scissors
1 ml syringe
15 ml sterile centrifuge tube
Centrifuge
37 °C, 5% CO2 cell culture incubator
Microscope
Hemocytometer
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lu, T. X. and Rothenberg, M. E. (2014). Bone Marrow Derived Eosinophil Cultures. Bio-protocol 4(12): e1161. DOI: 10.21769/BioProtoc.1161.
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Category
Immunology > Immune cell isolation > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell differentiation
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1,162 | https://bio-protocol.org/exchange/protocoldetail?id=1162&type=0 | # Bio-Protocol Content
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Peer-reviewed
Lysosomal Stability Assay
NP Nikolaj H. T. Petersen
TK Thomas Kirkegaard
MJ Marja Jäättelä
Published: Vol 4, Iss 12, Jun 20, 2014
DOI: 10.21769/BioProtoc.1162 Views: 16951
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Sep 2013
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Abstract
This assay makes use of the dye Acridine Orange (AO) to determine the stability of lysosomes in living cells upon exposure to a confocal microscope laser.
AO is a lipophilic amine that readily diffuses into cells (Figure 1). Inside the cell it enters the acidic lysosomal compartment where it is protonated and sequestered, shifting its emission spectrum towards a longer wavelength (i.e. red). Once inside the lysosomes, the metachromatic AO sensitizes the lysosomal membrane to photo-oxidation by blue light (Brunk et al., 1997). Upon light-induced loss of the lysosomal pH gradient and subsequent leakage of AO into the cytosol, the emission spectrum of AO shifts from red to green (Figure 2). Hence, loss of lysosomal integrity can be measured as a ‘loss of red dots’ or as a quantitative rise in green fluorescence (Petersen et al., 2010; Kirkegaard et al., 2010; Petersen et al., 2013).
Figure 1. Acridine Orange
Figure 2. Snapshots visualizing the U2OS cells at various steps of the recording procedure (Petersen et al., 2010)
Materials and Reagents
Cells (such as U-2 OS, ATCC, catalog number: HTB-96)
Growth medium (such as RPMI 1640 with 6% serum, Life Technologies, Gibco®)
Acridine Orange (Sigma-Aldrich, catalog number: 235474)
PBS containing 3% serum
Equipment
Zeiss Live DUO 5 confocal microscope or any newer microscope model capable of recording confocal images at high speed (500 ms/exposure), 100 mW diode laser, 40x c-apochromat objective
Lab-Tek 4 well (Nunc®, catalog number: 155383 ) or 8 well (Nunc®, catalog number: 155411 ) borosilicate cell chambers
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Petersen, N. H. T., Kirkegaard, T. and Jäättelä, M. (2014). Lysosomal Stability Assay. Bio-protocol 4(12): e1162. DOI: 10.21769/BioProtoc.1162.
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Category
Cancer Biology > General technique > Cell biology assays
Cell Biology > Cell staining > Organelle
Cell Biology > Cell imaging > Live-cell imaging
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1,163 | https://bio-protocol.org/exchange/protocoldetail?id=1163&type=0 | # Bio-Protocol Content
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Peer-reviewed
Phagolysosomal Trafficking Assay
Alka Mehra
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1163 Views: 15190
Reviewed by: Hong-guang XiaKanika Gera
Original Research Article:
The authors used this protocol in Oct 2013
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The authors used this protocol in:
Oct 2013
Abstract
Phagolysosomal trafficking is an important innate defense pathway that clears microbes by delivering them to lysosomes, the degradative compartment of the cell. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, subverts this host defense mechanism by arresting maturation of the phagosome. The ability of Mtb to arrest its delivery to the lysosome can be demonstrated by the prolonged co-localization of bacteria containing phagosomes/vacuole with early phagosomal markers [such as, Ras- related proteins in the brain 5 (Rab5) and Transferrin receptor (TfR)], and a failure to acquire late phagosomal and lysosomal markers (such as Rab7 and LAMP1) (Deretic and Fratti, 1999, Mehra et al., 2013). Here, a protocol is outlined for infection of macrophages with mycobacterial species like pathogenic Mtb, vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) and rapidly dividing non-pathogenic Mycobacterium smegmatis (Msmeg), followed by indirect-immunofluorescence microscopy to visualize host vacuolar markers. Thereafter, automated quantification of degree of co-localization between mycobacteria and host vacuolar markers like TfR and LAMP1 is done by processing the binary images of bacteria using mathematical tools. This results in quantification of the mean fluorescence intensity (MFI) of these host markers directly around the bacteria/bacterial clusters with increased sensitivity relative to when done manually. By manipulating host or pathogen, this assay can be used to evaluate host or bacterial determinants of intracellular trafficking. The basic method can be applied to studying trafficking of other bacteria or particles like beads, although the kinetics of infection and phagosome maturation will depend upon the phagocytic cargo. The mathematical analysis tools are available in many standard imaging analysis programs. However, any adaption for similar analysis should be confirmed by the individual user with their imaging and analysis platform.
Keywords: Macrophage infections Vacuolar staining Trafficking of bacteria Phagosomal fluorescence analysis Morphology and boolean logic operators
Materials and Reagents
Note: All work with live Mtb must be performed in a Biosafety Level 3 (BSL3) facility according to institutional standards of practice.
Macrophages, either primary macrophages, such as C57BL/6 bone marrow-derived macrophages (BMDMs) or a macrophage cell line (such as RAW264.7)
Note: BMDMs can be isolated as described (Banaiee et al. 2006; Nagabhushanam et el., 2013). RAW264.7 cells can be purchased from ATCC (ATCC, catalog number: TIB-71 ).
L929 cells (ATCC, catalog number: CCL-1 )
Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco®, catalog number: 11965 )
Fetal Bovine Serum (FBS) (heat inactivated) (Life Technologies, Gibco®, catalog number: 10082147 )
1 M HEPES solution (Life Technologies, Gibco®, catalog number: 15630-056 )
200 mM L-glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
Penicillin-Streptomycin solution (10,000 U/ml) (Life Technologies, Gibco®, catalog number: 15140-122 )
Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 10010-023 )
Eight well Permanox chamber slide (Thermo Fisher Scientific, Nunc Lab-Tek Chamber Slides, catalog number: 177445 )
Eight well chamber coverglass (Thermo Fisher Scientific, Nunc Lab-Tek Chamber coverglass, catalog number: 155411 )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
Bovine serum albumin (BSA) (fraction V) (Thermo Fisher Scientific, catalog number: BP1600 )
Detergents: Saponin (Sigma-Aldrich, catalog number: 47036 ), Triton- X100 (Sigma-Aldrich, catalog number: X100) and/or Tween-20 (Thermo Fisher Scientific, catalog number: BP337 )
Primary antibodies to detect host cellular markers
For example, recycling endosomes and early phagosomes can be labeled with mouse anti-transferrin receptor (anti-TfR) antibody (Life Technologies, InvitrogenTM, catalog number: 136800 ); Late endosomes and lysosomes stain with rabbit anti-LAMP1 antibody (Abcam, catalog number: 24170 ).
Notes:
If Mtb infected slides are to be removed from the BSL3 for imaging, the antibodies chosen need to work after fixation cum sterilization methods like long fixation as mentioned below in step A11, note a. Some of the commercially available antibodies may loose recognition or weakly recognize their epitopes after long fixation.
It is critical that polyclonal antibodies were not raised in animals given Freund’s adjuvant, as then they will directly recognize Mtb in addition to whatever cellular marker they were raised against. All antibodies should be tested to verify that they do not directly recognize Mtb.
Secondary antibodies for immunofluorescence
Secondary antibodies are available against different species and in different colors and user may choose depending on the primary antibodies being used. They are adsorbed against multiple species to minimize species cross reactivity during immunostaining. For example, Goat anti- mouse Alexa 594 (Life Technologies, Molecular Probes®, catalog number: A11032) and Goat anti-rabbit Alexa 594 (Life Technologies, Molecular Probes®, catalog number: A11037 ).
Of note, Mtb exhibits autofluorescence, with an emission maximum at 475 nm when excited at 405 nm, and thus are visualized by many DAPI filters (Patiño et al., 2008). Therefore, secondary antibodies should be chosen that do not fluoresce in this range.
Lysotracker Red DND-99 (1 mM stock in DMSO) (Life Technologies, Molecular Probes®, catalog number: L-7528 )
Note: Lysotracker dyes are available in different colors and one may choose depending on the color requirement.
Dextran (TexasRed, 10,000 MW, Lysine fixable) (Life Technologies, Molecular Probes®, catalog number: D-1863 ) (make 25 mg/ml stock in PBS, stored in dark in -20 °C)
Note: Dextran is available in different colors and molecular weights and again one may choose depending on requirement and desired goals of the experiment.
Vectashield mounting media (Vector Laboratories, catalog number: H-1000 )
Middle brook 7H9 broth (Difco, catalog number: 271310 )
Albumin-dextrose-catalase (ADC) (BD, catalog number: 212352 )
Oleic-albumin-dextrose-catalase (OADC) (BD, catalog number: 212351 )
Nail polish (clear)
Immersion oil (Microscope 50CC Immersion oil) (e.g. Nikon Corporation, catalog number: IB-MA-MXA20234 )
4% paraformaldehyde solution in PBS (see Recipes)
DMEM complete media (see Recipes)
DMEM/L929 complete media (see Recipes)
L-Cell conditioned media (see Recipes)
2% BSA in PBS containing 0.1% saponin (see Recipes)
2% BSA in PBS containing 0.1% triton X-100 (see Recipes)
7H9 complete media (see Recipes)
Fixative (see Recipes)
Blocking solution (see Recipes)
Equipment
Spectrophotometer (measure the OD600 i.e. optical density at wavelength of 600 nm of the mycobacterial cultures using cuvettes)
Disposable 1.5 ml cuvettes (Perfector Scientific, catalog number: 9003 )
Disposable sterile filter system (500 ml, 0.22 µm pore size) (Corning, catalog number: 430758 )
30 ml square media bottles (Thermo Fisher Scientific, Nalgene, catalog number: NE/2019-0030 )
50 ml, 15 ml falcon tubes (with plug seal caps) (Corning, catalog numbers: 430052 and 430290 )
Coverslip (22 x 50 mm, thickness#1, 0.13-0.17 mm) (Thermo Fisher Scientific, catalog number: 12-545C )
Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Beckman Coulter, model: Allegra X-15R ; bench top centrifuge with SX4750 rotor)
Note: Mtb cultures should be handled in BSL3 facility according to institutional standards of practice.
37 °C shaker incubator with aerosol containment units for Mtb liquid cultures
Beckman aerosolve canisters for centrifuging mycobacterial cultures in falcon tubes (e.g. Beckman Coulter, catalog number: BK359232 )
Multiwell-Plate Carrier Covers (e.g. Beckman Coulter, more on this link https://www.beckmancoulter.com/wsrportal/techdocs?docname=GX-TB-012)
37 °C shaker incubator with aerosol containment units for Mtb liquid cultures
Epifluorescence microscope [e.g. Nikon Eclipse TiE/B model equipped with 60x; Plan-Apochromat, NA 1.4 oil immersion objective, Ti Z drive, high resolution monochrome charge-coupled device (CCD) digital camera; Photometric Cool SNAP HQ2 and appropriate filter sets for DAPI, FITC and TexasRed channel]
Software
Nikon Imaging Software-Elements Advanced Research (NIS-Elements AR) version 3.2 software with deconvolution module
Graph Pad Prism software
Procedure
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Copyright: © 2014 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:
Mehra, A. (2014). Phagolysosomal Trafficking Assay . Bio-protocol 4(13): e1163. DOI: 10.21769/BioProtoc.1163.
Mehra, A., Zahra, A., Thompson, V., Sirisaengtaksin, N., Wells, A., Porto, M., Koster, S., Penberthy, K., Kubota, Y., Dricot, A., Rogan, D., Vidal, M., Hill, D. E., Bean, A. J. and Philips, J. A. (2013). Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog 9(10): e1003734.
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Category
Immunology > Immune cell function > Macrophage
Cell Biology > Cell imaging > Fluorescence
Microbiology > Microbe-host interactions > In vitro model
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1,164 | https://bio-protocol.org/exchange/protocoldetail?id=1164&type=0 | # Bio-Protocol Content
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Peer-reviewed
Grafting Arabidopsis
TA Tonni Grube Andersen
DL Dacheng Liang
BH Barbara Ann Halkier
RW Rosemary White
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1164 Views: 16249
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Aug 2013
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Aug 2013
Abstract
In Arabidopsis thaliana, hypocotyl micrografting has been used to investigate transport of flowering signals, mobile silencing signals and other peptides, proteins and secondary compounds. The effects of transported signals on target tissues require that a good vascular connection is re-established across the graft junction between the cut hypocotyls (stumps) of the root (rootstock) and shoot (scion) tissues. We outline here a method that requires only that the cut stumps be placed in close proximity, so that they touch, followed by 3-5 days of undisturbed recovery time during which the grafts are allowed to dry out somewhat. This method is quick, easy to monitor and has up to 90% success rate.
Keywords: Grafting Arabidopsis Hypocotyl Rootstock Scion
Materials and Reagents
Young Arabidopsis seedlings (grown for 3-6 days on ½ MS containing agar plates)
Sterile distilled water
96 % ethanol
Equipment
Sterile 9 cm petri dishes
Sterile standard Whatman No. 1 filter paper circles Grade 1:11 µm with diameter about 0.5 cm smaller than the sterile petri dish (autoclaved wrapped in foil beforehand) (8.5 cm diameter) (Whatman)
Sterile cellulose nitrate filters (Whatman, catalog number: NC 45 ST)
Sterile forceps
Sterile 1 ml pipette tips
1 ml pipettor
Micropore tape (3 M, MicroporeTM)
Sapphire knife holder (World Precision Instruments, catalog number: 500317 ) or diamond knife plus blade (Electron Microscopy Sciences, Diamond Dissecting Knife, Type M-M, catalog number: 72025 )
Sapphire blade (World Precision Instruments, catalog number: 504072 ) or diamond blade (Electron Microscopy Sciences, Blade Resharpening, Type M-M, catalog number: 72025-R ; Electron Microscopy Sciences, Replacement Blade, Type M-M, catalog number: 72025-L )
Flow cabinet mounted dissecting microscope with a minimum of 5x magnification
Note: Any brand we have tried worked.
Parafilm
Hot glass bead sterilizer (any brand) unless using 96 % ethanol for sterilization
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Andersen, T. G., Liang, D., Halkier, B. A. and White, R. (2014). Grafting Arabidopsis. Bio-protocol 4(13): e1164. DOI: 10.21769/BioProtoc.1164.
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Category
Plant Science > Plant physiology > Plant growth
Plant Science > Plant developmental biology > Morphogenesis
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1,165 | https://bio-protocol.org/exchange/protocoldetail?id=1165&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analyses of Plant Leaf Cell Size, Density and Number, as Well as Trichome Number Using Cell Counter Plugin
YC Yan Cheng
LC Ling Cao
SW Sheng Wang
YL Yongpeng Li
HW Hong Wang
YZ Yongming Zhou
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1165 Views: 26010
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Aug 2013
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Aug 2013
Abstract
An Arabidopsis leaf blade is composed of many layers that are sandwiched between two layers of tough skin cells (called the epidermis). Four layers (adaxial epidermis, palisade layer, spongy mesophyll and abaxial epidermis) contain specialized cells. Here we describe a quick and simple method for analyzing the size, number and density of different types of cells in an Arabidopsis leaf blade. This method would be of interest to people who would like to investigate cell size and number changs in different cell layers in leaves or leaf-like organs without having to dissect the samples.
Materials and Reagents
Dissected rosette leaves from Arabidopsis
Petals from the well-opened flower
Ethanol (Sigma-Aldrich, Catalog number: 459844 )
Chloral hydrate (Sigma-Aldrich, Catalog number: 15307 )
Glycerol (Sigma-Aldrich, Catalog number: G7757 )
Washing solution (see Recipes)
Clearing solution (see Recipes)
Equipment
Stereomicroscope (Zeiss, model: Zeiss Stemi 2000 )
Digital Cameral (Canon, model: Canon powershot S5IS )
Glass slides and thin coverslips
Differential interference contrast (DIC) microscope (Nikon Corporation, model: ECLIPSE 80i) with CCD camera (Nikon Corporation, model: DS-Ri1 )
Software
Image J software (http://rsbweb.nih.gov/ij/index.html).
Cell Counter plugin (http://rsbweb.nih.gov/ij/plugins/cell-counter.html)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant cell biology > Cell structure
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Live-cell imaging
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1,166 | https://bio-protocol.org/exchange/protocoldetail?id=1166&type=0 | # Bio-Protocol Content
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Peer-reviewed
Plant Sequence Capture Optimised for Illumina Sequencing
Axel Himmelbach
Manuela Knauft
NS Nils Stein
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1166 Views: 15099
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Nov 2013
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Abstract
Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole exome of barley. The Exome Library is provided as a liquid array containing biotinylated probes (Roche/NimbleGen). Subsequently, genomic shotgun DNA fragments hybridized to the Exome Library are affinity-purified using streptavidin coated magnetic beads. The captured library is PCR-amplified and sequenced using high-throughput short read sequencing-by-synthesis.
Keywords: Sequence capture Targeted resequencing Exome Sequencing-by-synthesis Barley
Materials and Reagents
SYBR Gold (Life Technologies, catalog number: S11494 )
UltraPure Agarose (Life Technologies, InvitrogenTM, catalog number: 16500-500 )
PCR-grade water
Tris (hydroxymethyl) aminomethane (Tris base) (multiple vendors)
Tween 20 (Bio-Rad Laboratories, catalog number: 170-6531 )
Ethylenediaminetetraacetic acid (EDTA) (multiple vendors)
Ethanol (absolute) (analytical grade) (multiple vendors)
70 % (v/v) ethanol (analytical grade)
Isopropanol (2-Propanol) (>99.5%) (multiple vendors)
Acetic acid (glacial) (multiple vendors)
2x Phusion High-Fidelity PCR Master Mix (New England BioLabs, catalog number: F-531L )
GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific, catalog number: SM 0371 )
QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
Minelute PCR Purification Kit (QIAGEN, catalog number: 28006 )
Agilent DNA 7500 Kit (Agilent Technologies, catalog number: 5067-1506 )
Agilent High Sensitivity DNA Kit (Agilent, catalog number: 5067-4626 )
SeqCap EZ Hybridization Kit [containing NimbleGen SC Wash Buffers (tubes 1, 2 and 3), Stringent Wash Buffer (tube 4), 2x SC Hybridisation Buffer (tube 5), Hybridisation Component A (tube 6) and the Bead Wash Buffer (tube 7)] (Roche Diagnostics, catalog number: 05634261001 )
Sequence Capture Developer Reagent (Roche Diagnostics, catalog number: 06684335001 )
Note: This reagent was previously known as Plant Capture Enhancer (PCE) from Roche NimbleGen.
6x loading dye (Thermo Fisher Scientific, Fermentas, catalog number: R 0611 )
DNA away (Thermo Fisher Scientific, catalog number: 21-236-28 )
Oligonucleotides (Sigma-Aldrich)
Note: All oligonucleotides were reverse phase cartridge purified and dissolved in PCR-grade water. To avoid cross-contaminations each oligonucleotide was purified using a fresh column. Sequences are listed in Table 1 in Supplementary Material.
Reagents for the generation of capture libraries:
Illumina TruSeq DNA kit (Illumina, catalog number: FC-121-2001 , Box A) or
TruSeq DNA PCR-free sample preparation kit (Illumina, catalog number: FC-121-3001 , Set A)
If the DNA Illumina multiplex (IM) protocol is used, the following additional reagents (items 27- 41) are required.
T4 polynucleotide kinase (10 U/µl) (Thermo Scientific Fermentas, catalog number: EK0032 )
T4 DNA polymerase (5 U/µl) (Thermo Scientific Fermentas, catalog number: EP006B )
10x Buffer Tango (Thermo Scientific Fermentas, catalog number: BY5 )
dNTPs (25 nM each) (Thermo Scientific Fermentas, catalog number: R1121 )
ATP (100 mM) (Thermo Fisher Scientific, catalog number: R0441 )
T4 DNA ligase (5 U/µl) provided with 10x T4 DNA ligase buffer and 50% PEG 4000 (Thermo Scientific Fermentas, catalog number: EL0011 )
10x ThermoPol reaction buffer (New England Biolabs, catalog number: B9004S )
Bst polymerase (8 U/µl) (large fragment) (New England Biolabs, catalog number: M0275L )
5x Phusion HF buffer (New England Biolabs, catalog number: B0518S )
Phusion Hot Start Flex DNA polymerase (2 U/µl) (New England Biolabs, catalog number: M0535L )
carboxyl-modified Sera-Mag Magnetic Speed-beads (Thermo Fisher Scientific, catalog number: 1182-9912 )
PEG-8000 (Sigma-Aldrich, catalog number: 89510 )
NaCl (Sigma-Aldrich, catalog number: S3014 )
Agilent High Sensitivity DNA Kit (Agilent, catalog number: 5067-4626)
Agilent DNA 7500 Kit (Agilent, catalog number: 5067-1506)
TE (pH 8.0) (see Recipes)
Resuspension Buffer (RSB) (see Recipes)
50x TAE (see Recipes)
EBT (see Recipes)
Test fragment for DNA Illumina multiplex (IM) libraries (see Recipes)
Adapter mix P57 for IM libraries (see Recipes)
MagNA beads for DNA clean-up (see Recipes)
Equipment
Qubit 2.0 fluorometer (Starter Kit) [including instrument, assay tubes, dsDNA HS Assay and dsDNA BR Assay] (Thermo Fisher Scientific, catalog number: Q32871 ) or other picogreen-based dsDNA quantification devices
AMPure XP Beads (Beckman Coulter, catalog number: A63882 )
Streptavidin Dynabeads (M-270) (Life Technologies, InvitrogenTM, catalog number: 65306 )
1.5 ml tubes (multiple vendors)
0.2 ml PCR-tubes (multiple vendors)
96-well plates (Greiner Bio-One GmbH, catalog number: 652250 )
Plastic seals for 96 well plate (Thermo Fisher Scientific, catalog number: AB-0558 )
Covaris S220 AFA Ultrasonicator (LGC, catalog number: KBS-500217 ) and associated equipment such as microTUBE holder, chiller, software, computer etc.
Snap-Cap microTUBES with AFA-fiber and pre-split septum (Covaris, catalog number: 520045 )
Agilent 2100 Electrophoresis Bioanalyzer (Agilent, catalog number: G2939AA ) and associated material (e.g. computer)
NanoDrop 2000 Spectrophotometer (VWR International, PeQlab, catalog number: 91-ND-2000 )
HiSeq or MiSeq Illumina systems and associated materials (such as Sequencing-by-Synthesis reagents for 2x 100 cycles)
Heating block (multiple vendors)
Water Bath with external calibrated thermometer (multiple vendors)
Microcentrifuge (16,000 x g) (multiple vendors)
SpeedVac (multiple vendors)
Thermocycler (multiple vendors)
Note: Throughout the protocol a “Bio Rad DNA Engine Tetrad 2 Peltier Thermal Cycler” was used.
Vortex Mixer (multiple vendors)
DynaMag-2 Magnet (Life Technologies, InvitrogenTM, catalog number: 123-21D )
DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) (Life Technologies, InvitrogenTM, catalog number: 120.27 )
Agarose gel electrophoresis equipment and accessories [microwave, tray (15 x 15 cm), combs, power supply, UV-transilluminator, etc.] (multiple vendors)
Disposable scalpels (multiple vendors)
Dark Reader blue light transilluminator (Clare Chemical Research, catalog number: DR46B )
Pipettes (2, 100, 200 and 1,000 µl, multiple vendors)
Filter tips (multiple vendors)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Himmelbach, A., Knauft, M. and Stein, N. (2014). Plant Sequence Capture Optimised for Illumina Sequencing. Bio-protocol 4(13): e1166. DOI: 10.21769/BioProtoc.1166.
Download Citation in RIS Format
Category
Plant Science > Plant molecular biology > DNA
Systems Biology > Genomics > Exome capture
Molecular Biology > DNA > DNA labeling
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1,167 | https://bio-protocol.org/exchange/protocoldetail?id=1167&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Cytokinin Analysis: Sample Preparation and Quantification
Martin Schäfer
MR Michael Reichelt
IB Ian T. Baldwin
SM Stefan Meldau
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1167 Views: 15202
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Nov 2013
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Nov 2013
Abstract
Cytokinins are a group of phytohormones discovered about half a decade ago by Miller et al. (1955) and Skoog et al. (1965). Since then they were found to participate in many plant physiological processes, including the regulation of the source/sink transitions, plant growth and organ development, responses to environmental conditions such as light, nutrient and water availability and biotic interactions with mutualists, pathogens and herbivores (Werner and Schmülling, 2009; Giron et al., 2013). To aid the quantification of cytokinins for analyzing their changes after environmental stress conditions, we developed this cytokinin extraction and analysis method. This protocol is based on the cytokinin extraction with an acidic methanol-water solution and purification with a mixed-mode solid phase extraction procedure described by Dobrev and Kamı́nek (2002) and the modifications of Kojima et al. (2009). The protocol was successfully used to verify cytokinin overproduction in transgenic Nicotiana attenuata plants expressing the cytokinin biosynthesis gene Tumor morphology root (Tmr) from Agrobacterium tumefaciens under the control of the chemical inducible expression system pOp6/LhGR in the glasshouse and under field conditions (Schäfer et al., 2013) to study the role of cytokinins in plant-herbivore interactions.
Materials and Reagents
Plant tissue
MeOH
1 N HCOOH
0.35 N NH4OH
0.35 N NH4OH in 60% MeOH
0.1% (v/v) acetic acid
0.05% (v/v) HCOOH (for mass spectrometry) in Milli-Q H2O
Acetonitrile (gradient grade)
[2H5] tZ (Olchemim, catalog number: 030 0301 )
[2H5] tZR (Olchemim, catalog number: 030 0311 )
[2H5] tZROG (Olchemim, catalog number: 030 5131 )
[2H5] tZ7G (Olchemim, catalog number: 030 5111 )
Extraction buffer (see Recipes)
Extraction buffer + deuterated standards (see Recipes)
Equipment
96-well BioTubes (1.1 ml individual tubes) (Arctic White LLC, catalog number: AWTS-X22100 )
Steel balls (ASKUBAL, catalog number: 3 MM-G100-1.4034 )
Caps for 96-well BioTubes (strips of 8 plug caps) (Arctic White LLC, catalog number: AWSM-T100-30 )
Pipet
ArctiSeal 96 Round Well Sealing Mats for 96-well BioTubes (Arctic White LLC, catalog number: AWSM-2002RB )
Nunc 96-well Deep Well Plates (Thermo Fisher Scientific, catalog number: 278752 )
Nunc 96-Well Cap Mats (Thermo Fisher Scientific, catalog number: 276002 )
96-well PCR plates (Kaneka Corporation, Eurogentec, catalog number: RT-PL96-MQ )
Sealing film (OMNILAB-LABORZENTRUM, Schubert & Weiss, catalog number: 5420203 )
Machery Nagel Multi 96 HR-X (96 x 25 mg) (MACHEREY-NAGEL, catalog number: 738530.025M )
Machery Nagel Multi 96 HR-XC (96 x 25 mg) (MACHEREY-NAGEL, catalog number: 738540.025M )
Chromabond Multi 96 vacuum manifold (MACHEREY-NAGEL, catalog number: 738630.M )
Evaporator system (Glas-Col, catalog number: 099A EV9624S )
Geno/Grinder 2000 (SPEX SamplePrep)
Eppendorf Centrifuge 5804 R equipped with a Swing-bucket rotor A-2-DWP (Eppendorf)
Ultrasonic bath Bransonic Models 1200 (BRANDSONTM)
Agilent 1200 HPLC system (Agilent)
Zorbax Eclipse XDB-C18 column (50 x 4.6 mm, 1.8 µm) (Agilent)
API 5000 tandem mass spectrometer (Applied Biosystems®) equipped with a Turbospray ion source
Procedure
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Category
Plant Science > Plant biochemistry > Plant hormone
Plant Science > Plant physiology > Tissue analysis
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1,168 | https://bio-protocol.org/exchange/protocoldetail?id=1168&type=0 | # Bio-Protocol Content
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Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria
LL Liang-Dong Lyu
GZ Guo-Ping Zhao
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1168 Views: 11596
Original Research Article:
The authors used this protocol in Dec 2013
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Abstract
Understanding the genetic safeguarding mechanism of Mycobacterium tuberculosis (Mtb) may help us to explain i), how Mtb survive the genetic assaults elicited by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host macrophages and ii), why some strains of Mtb, e.g., Mtb strains from East Asian lineage and Beijing sublineage, exhibit high mutation rate and are more likely to acquire drug resistant mutations (e.g., rifampicin-resistance mutation) during infection. Mutation frequency analysis is a basic methods to study the genetic safeguarding mechanism. Moreover, to study the molecular mechanism of mutation, it is necessary to analyse the mutation spectrum (For example, oxidized cytosine may induce CG to TA mutation.). This protocol describes a method to determine the mutation frequency and understand the mutation spectrum in both Mycobacterium smegmatis (Msm) and Mtb.
Keywords: Mycobacterium Mutation frequency DNA damage
Materials and Reagents
Mycobacterium smegmatis mc2 155 or Mycobacterium tuberculosis H37Rv
Middlebrook 7H9 broth (BD Biosciences, catalog number: 271310 )
Middlebrook 7H11 agar (BD Biosciences, catalog number: 283810 )
Middlebrook OADC (BD Biosciences, catalog number: 212351 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Tween 80 (Sigma-Aldrich, catalog number: P1754 )
Rifampicin (Sigma-Aldrich, catalog number: R3501 )
7H9 medium (see Recipes)
7H9OADC medium (see Recipes)
7H11 agar (see Recipes)
7H11OADC agar (see Recipes)
LBG agar (see Recipes)
PBST (see Recipes)
TE buffer (see Recipes)
Equipment
Incubation shaker
Centrifuge
37 °C incubator
100-ml flask
Sterile glass beads
96-well flat bottom plate
Adhesive film
96-well PCR plate
PCR instrument
Petri dish (70 mm diameter)
Software
BLAST or ClustalW
Procedure
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Copyright: © 2014 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:
Lyu, L. and Zhao, G. (2014). Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria. Bio-protocol 4(13): e1168. DOI: 10.21769/BioProtoc.1168.
Lyu, L. D., Tang, B. K., Fan, X. Y., Ma, H. and Zhao, G. P. (2013). Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP. PLoS Pathog 9(12): e1003814.
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Category
Microbiology > Microbial genetics > Mutagenesis
Microbiology > Microbial cell biology > Cell isolation and culture
Molecular Biology > DNA > Mutagenesis
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1,169 | https://bio-protocol.org/exchange/protocoldetail?id=1169&type=0 | # Bio-Protocol Content
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Peer-reviewed
Extraction of Nonstructural Carbon and Cellulose from Wood for Radiocarbon Analysis
CC Claudia I. Czimczik
ST Susan E. Trumbore
XX Xiaomei Xu
MC Mariah S. Carbone
AR Andrew D. Richardson
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1169 Views: 8499
Edited by: Tie Liu
Reviewed by: Masahiro Morita
Original Research Article:
The authors used this protocol in Feb 2013
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Abstract
This method aims at isolating nonstructural organic carbon (NSC) pools, i.e. soluble sugars and starch, from wood for radiocarbon (14C) analysis at natural abundance levels (≤1 ppt).
Pools are operationally defined to 1) physically isolate pools - prohibiting the use of destructive methods, such as compound-specific enzyme digestion, and 2) minimize possible contamination with extraneous carbon form organic solvents.
Materials and Reagents
Nonstructural carbon and cellulose
MQ H2O
Laboratory grade pure methanol for cleaning
Nonstructural carbon
14C standards [international secondary standards: ANU sucrose (IAEA-C6), in-house laboratory standards, e.g. wheat flour, rye flour]
Laboratory-grade ethanol
HPLC-grade ethanol
Analytical-grade 20% HCl [≈ 6 M HCl, 50:50 (v: v) conc. HCl]
Cellulose
14C standards (international secondary wood standards: e.g. IAEA-C4, -C5)
Laboratory-grade toluene
Laboratory-grade glacial acetic acid (CH3COOH)
Technical grade sodium chlorite (NaClO2)
Equipment
Nonstructural carbon and cellulose
Aluminum (Al) foil (cover surfaces and prevent dust contaminating equipment and samples with extraneous carbon)
Marker pen
Spatula
Wipes for cleaning (e.g. KCWW)
Duster for cleaning (I L) (e.g. InnoveraTM)
Pyrex beakers to hold reagents
Graduated volumetric cylinder to prepare reagents
Note: All glassware is baked and stored covered with Al foil to remove extraneous sources of carbon. Quartz is pre-combusted at 900 °C for 2 h, Pyrex at 550 °C for 3 h.
Nonstructural carbon
Mill with mesh 10 or scalpel
Graduated, adjustable volume, 5 ml pipette (e.g. VWR International, Eppendorf) or cylinder
Disposable borosilicate glass culture tubes (13 x 100 mm) with plastic vented caps and snap caps (VWR International)
Disposable, plastic (extended fine tip) transfer pipette (e.g. Thermo Fisher Scientific, Samco Scientific)
Quartz combustion tubes (120 mm length, 9 mm O.D.)
50 ml reusable glass centrifugation tube with screw cap or plastic (e.g. PP) centrifuge tubes with screw cap
Speedvac-dryer (Thermo Fisher Scientific, Savant, model: SC200 speedvac with RT400 cold trap) (operated at room temperature)
Block heater with thermometer for 10 or 20 (13 x 100 mm) disposable borosilicate glass culture tubes
Cellulose
General-purpose extra-long, stainless steel forceps
Quartz combustion tubes (150 mm length, 6 mm O.D.)
Heat sealer
1,000 ml pyrex beaker
1,000 ml pyrex round bottom flask (still pot)
Soxhlet extraction apparatus (e.g. VWR International)
Heated magnetic stirrer & stir bar
Drying oven
Cork ring
Boiling chips
Fiber filter bags for sample digestion (ANKOM Technology, catalog number: F57 )
Procedure
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How to cite：Czimczik, C. I., Trumbore, S. E., Xu, X., Carbone, M. S. and Richardson, A. D. (2014). Extraction of Nonstructural Carbon and Cellulose from Wood for Radiocarbon Analysis. Bio-protocol 4(13): e1169. DOI: 10.21769/BioProtoc.1169.
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Category
Plant Science > Plant biochemistry > Carbohydrate
Biochemistry > Carbohydrate > Polysaccharide
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1,170 | https://bio-protocol.org/exchange/protocoldetail?id=1170&type=0 | # Bio-Protocol Content
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Pulse Chase of Suspension Cells
Lai-Yee Wong
QiMing Liang
Kevin Brulois
Jae Jung
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1170 Views: 12196
Original Research Article:
The authors used this protocol in Nov 2013
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Abstract
Pulse-chase method is a powerful technique used to follow the dynamics of proteins over a period of time. The expression level, processing, transport, secretion or half-life of proteins can be tracked by metabolically labeling the cells, such as with radiolabeled amino acids (pulse step). This protocol describes the condition used to study the folding and disulfide bond formation of immunoglobulin in suspension cells. With some minor modifications, this protocol can be adapted to study the degradation rate or the secretion of target proteins.
Keywords: Protein folding Protein degradation Protein secretion
Materials and Reagents
Pulse chase
Cells growing in suspension
HBSS (Life Technologies, InvitrogenTM, catalog number: 14175-095 )
RPMI without methionine and cysteine (Sigma-Aldrich, catalog number: R7513 )
Dialyzed FBS (Life Technologies, InvitrogenTM, catalog number: 26400044 )
N-Ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: R3876 )
Cyclohexamide (CHX) (Sigma-Aldrich, catalog number: C7698 )
Express 35S protein labelling mix (Perkin Elmer, catalog number: NEG072014MC )
Methionine (Sigma-Aldrich, catalog number: M5308 )
Cysteine (Sigma-Aldrich, catalog number: C7352 )
Labeling medium (see Recipes)
Chase medium (see Recipes)
2x stop buffer (see Recipes)
Cell lysis and immunoprecipitation
Antibody against protein of interest
Protein A/G beads (Thermo Fisher Scientific, catalog number: 20422 )
Complete Protease Inhibitor Tablets (Roche Diagnostics, catalog number: 11836145001 )
Lysis buffer (see Recipes)
SDS-PAGE
4-12% Bis-Tris protein gel (Life Technologies, InvitrogenTM)
MOPS running buffer (Life Technologies, InvitrogenTM, catalog number: NP0001 )
Amplify solution (GE Healthcare, catalog number: NAMP100 )
Gel drying solution (Life Technologies, InvitrogenTM, catalog number: LC4025 )
Gel fixing solution (see Recipes)
Equipment
Incubator
Eppendorf tube
26-gauge needle
1 ml syringe
Heat block
Gel dryer
Phosphor imaging screen
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wong, L., Liang, Q., Brulois, K. and Jung, J. (2014). Pulse Chase of Suspension Cells. Bio-protocol 4(13): e1170. DOI: 10.21769/BioProtoc.1170.
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Category
Immunology > Antibody analysis > Antibody detection
Biochemistry > Protein > Labeling
Biochemistry > Protein > Structure
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1,171 | https://bio-protocol.org/exchange/protocoldetail?id=1171&type=0 | # Bio-Protocol Content
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Peer-reviewed
Murine in vitro Memory T Cell Differentiation
MK Myoungjoo V. Kim
WO Weiming Ouyang
WL Will Liao
MZ Michael Q. Zhang
ML Ming O. Li
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1171 Views: 14165
Reviewed by: Omar Akil
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
Upon pathogen encounter, naïve CD8+ T cells are primed and undergo massive clonal expansion. A fraction of effector CD8+ T cells remains during the contraction phase and differentiate into memory T cells critical for mounting robust recall responses in response to secondary infection. Low frequency of memory T cells in vivo is a major obstacle to investigate their functional aspects including migration capacity and genetic regulation. Here, we describe detailed protocol for memory T cell differentiation developed by von Andrian’s group to generate large number of CD44hiCD62Lhi antigen-specific memory T cells in vitro.
Keywords: Ag-specific memory CD8 T cell OT-I IL-15 In vitro CD44+CD62L+ central memory T cell
Materials and Reagents
Recombinant mouse IL-15 (rmIL15) (BioLegend, catalog number: 566302 )
RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-119 )
Fetal bovine serum (Atlanta Biologicals, catalog number: S11055H )
Penicillin/streptomycin (Gemini Bio-Products, catalog number: F52M00E )
L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
100x 1 M Hepes (Life Technologies, Gibco®, catalog number: 15630-080 )
100x MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140-050 )
100x sodium pyruvate (100 mM) (Life Technologies, Gibco®, catalog number: 11360-070 )
100x 2-mercaptoethanol (Life Technologies, Gibco®, catalog number: 21985-023 )
OVA257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951 )
Ficoll-PaqueTM Premium 1.084 (GE Healthcare, catalog number: 17-5446-02 )
Antibodies:
Anti-CD44 PerCpCy5.5 (clone: IM7) (eBioscience, catalog number: 45-0441 )
Anti-CD62L APC (clone: MEL-14) (eBioscience, catalog number: 17-0621 )
RBC lysis buffer (eBioscience, catalog number: 00-4333-57 )
Bovine serum albumin (Thermo Fisher Scientific, catalog number: BP1605-100 )
NaN3 (Sigma-Aldrich, catalog number: S8032 )
T cell media (see Recipes)
Staining buffer (in PBS) (see Recipes)
Equipment
Centrifuge (Thermo Fischer Scientific, SorvallTM Legend RT)
70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
15 ml and 50ml Falcon tubes
24 well plates (BD Biosciences, Falcon®, catalog number: 353226 )
T75 culture flask (Corning, catalog number: 430641 )
37 °C, 5% CO2 cell culture incubator
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Immune cell function > Lymphocyte
Immunology > Immune cell function > Antigen-specific response
Immunology > Immune cell isolation > Maintenance and differentiation
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1,172 | https://bio-protocol.org/exchange/protocoldetail?id=1172&type=0 | # Bio-Protocol Content
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Peer-reviewed
Murine in vivo CD8+ T Cell Killing Assay
MK Myoungjoo V. Kim
WO Weiming Ouyang
WL Will Liao
MZ Michael Q. Zhang
ML Ming O. Li
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1172 Views: 18665
Reviewed by: Omar Akil
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
Antigen-specific killing ability of effector CD8+ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8+ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8+ T cell responses. Effector CD8+ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into the previously immunized mice. Difference in CFSE expression levels clearly marks two distinct populations: Antigen-pulsed target cells-CFSElow vs. unpulsed target cells-CFSEhi. The frequencies between antigen-pulsed target cells and control target cells are used as readouts of antigen-specific killing.
Materials and Reagents
Splenocytes from a wild type mouse
PBS (Thermo Fisher Scientific, catalog number: BP399-20 )
Note: 10x solution, diluted to 1x in house in distilled water and sterilized by autoclave.
RBC lysis buffer (eBioscience, catalog number: 00-4333-57 )
HBSS without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14175-095 )
RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-119 )
Fetal bovine Serum (Atlanta Biologicals, catalog number: S11055H )
Penicillin/streptomycin (Gemini Bio-Products, catalog number: F52M00E )
L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
Trypan blue solution (Life Technologies, Gibco®, catalog number: 15250-061 )
OVA257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951 )
5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich, catalog number: 21888 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D-8418 )
Collagenase D (Sigma-Aldrich, catalog number: C-5138 )
Percoll (Sigma-Aldrich, catalog number: P-1644 )
Complete RPMI-1640 media (see Recipes)
100% percoll solution (see Recipes)
Equipment
Centrifuge (Thermo Fischer Scientific, SorvallTM Legend RT )
37 °C water bath
Hemocytometer
15 ml and 50 ml Falcon tubes
6 well plates (USA Scientific, CytoOne®, catalog number: CC7682-7506 )
BD LSRII Flow Cytometer (BD)
70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
5 ml polystyrene round-bottom tubes with cell-strainer cap (BD Biosciences, Falcon®, catalog number: 352235 )
3 ml syringe (BD, catalog number: 14-823-435 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. and Li, M. O. (2014). Murine in vivo CD8+ T Cell Killing Assay. Bio-protocol 4(13): e1172. DOI: 10.21769/BioProtoc.1172.
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Category
Immunology > Immune cell function > Antigen-specific response
Immunology > Animal model > Mouse
Microbiology > Microbe-host interactions > In vivo model
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1,173 | https://bio-protocol.org/exchange/protocoldetail?id=1173&type=0 | # Bio-Protocol Content
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Peer-reviewed
Seed Coat Permeability Test: Tetrazolium Penetration Assay
Sollapura J. Vishwanath
Frédéric Domergue
Owen Rowland
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1173 Views: 13816
Edited by: Renate Weizbauer
Original Research Article:
The authors used this protocol in Nov 2013
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Nov 2013
Abstract
Seed coat permeability is important to study as it plays significant roles in seed dormancy, germination, and protection from pathogens. Here we describe a commonly used seed coat permeability test known as the tetrazolium penetration assay with a method to quantify the levels of permeability. Tetrazolium red is a cationic dye that is widely used in seed viability testing. Tetrazolium salts are amphipathic cations, which, after penetrating the dead cells of the seed coat, are reduced to red-colored insoluble precipitates made up of formazans by active dehydrogenases (NADH-dependent reductases) in the embryo of seeds (Berridge et al., 1996). The intensity of red coloration is directly proportional to the permeability of the seeds. The quantification involves extraction of formazans from the incubated seeds and spectrophotometric determination of absorbance of formazan extracts at 485 nm.
Note: This protocol is optimized for testing Arabidopsis thaliana seeds.
Keywords: Seed coat Permeability Tetrazolium Formazan Arabidopsis
Materials and Reagents
Seeds (50 mg)
2,3,5-triphenyltetrazolium chloride (tetrazolium red) (Sigma-Aldrich, catalog number: T8877 )
95% ethanol (spectrophotometric grade)
Distilled or ultra-pure deionized water
Equipment
Brown bottle
1.5 ml microcentrifuge tubes
Microcentrifuge tube rack
Aluminum foil
Air incubator (set to 30 °C)
1 ml Pipette (e.g., Gilson)
Pestle and mortar
Disposable pasteur pipettes (glass)
Spectrophotometer
Spectrophotometer cuvettes (polystyrene, 1.5 ml, path length 1 cm)
Microcentrifuge
Stereomicroscope with color camera
Procedure
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Copyright: © 2014 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:
Vishwanath, S. J., Domergue, F. and Rowland, O. (2014). Seed Coat Permeability Test: Tetrazolium Penetration Assay. Bio-protocol 4(13): e1173. DOI: 10.21769/BioProtoc.1173.
Vishwanath, S. J., Kosma, D. K., Pulsifer, I. P., Scandola, S., Pascal, S., Joubes, J., Dittrich-Domergue, F., Lessire, R., Rowland, O. and Domergue, F. (2013). Suberin-associated fatty alcohols in Arabidopsis: distributions in roots and contributions to seed coat barrier properties. Plant Physiol 163(3): 1118-1132.
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Category
Plant Science > Plant physiology > Tissue analysis
Plant Science > Plant cell biology > Cell staining
Cell Biology > Tissue analysis > Tissue staining
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1,174 | https://bio-protocol.org/exchange/protocoldetail?id=1174&type=0 | # Bio-Protocol Content
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Extraction of Ions from Leaf Sections
Yuko Kurita
Satomi Kanno
Miwa Ohnishi
Tetsuro Mimura
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1174 Views: 6936
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Oct 2013
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The authors used this protocol in:
Oct 2013
Abstract
The concentration of ions in plant cells and tissues is an important factor to determine their functions and conditions. Here, we describe the method to extract ions from leaf sections for measurements with an ion chromatogram. This method is available for not only barley but also other plant species.
Keywords: Ion chromatography Barley Inorganic ion
Materials and Reagents
Barley seedlings
Milli-Q water
Hydroponic culture solution (see Recipes)
Equipment
1.5 ml and 2 ml plastic tubes
Scissors and forceps
Sample crusher (Kurabo Industries, model: SH-48 )
7 mm zirconia beads (Kurabo Industries, model: Z-07 )
Vortex mixer
Dry thermo unit (Taitec, catalog number: DTU-18 )
0.45 µm filters (EMD Millipore, catalog number: SLLHH04NL )
1.0 ml syringe
Centrifuge
Ion chromatogram (Thermo Fisher Scientific, Dionex, model: ICS-1500 ) equipped with an ion exchange column (AS-12 for anions and CS-16 for cations)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kurita, Y., Kanno, S., Ohnishi, M. and Mimura, T. (2014). Extraction of Ions from Leaf Sections. Bio-protocol 4(13): e1174. DOI: 10.21769/BioProtoc.1174.
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Category
Plant Science > Plant physiology > Ion analysis
Biochemistry > Other compound > Ion
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1,175 | https://bio-protocol.org/exchange/protocoldetail?id=1175&type=0 | # Bio-Protocol Content
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Autoradiography of Pi Distribution in Barley Seedlings
Satomi Kanno
Yuko Kurita
Miwa Ohnishi
Tetsuro Mimura
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1175 Views: 8866
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Oct 2013
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The authors used this protocol in:
Oct 2013
Abstract
Phosphorus-32 and Phosphorus-33 are radioisotopes of phosphorus. These isotopes are used to trace ionic phosphorus and phosphorus compounds. This protocol is used to follow the movement of inorganic phosphate (PO43-) from a leaf tip to the rest of the plant.
Keywords: Autoradiogram Imaging plate Barley Phosphate Translocation
Materials and Reagents
Barley seedlings
Radioisotopes 32P or 33P labeled NaH2PO4 dissolved in water (MP Biomedicals, PerkinElmer or American Radiolabeled Chemicals)
5 mM CaSO4 solution
Hydroponic culture solution (see Recipes)
Equipment
Cling film
1.5 ml plastic tubes
15 ml plastic tubes (1.5 ml tube is fitted by opening a hole in the lid) (Figure 1)
Cotton
Plastic sponge
Imaging plate (FCR Imaging Plate for general purpose) (Fujifilm Corporation) and plate cassette (FCR standard cassette) (Fujifilm Corporation)
Imaging analyzer (GE Healthcare, model: Typhoon 9400 or other Radioisotope imaging analyzers)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kanno, S., Kurita, Y., Ohnishi, M. and Mimura, T. (2014). Autoradiography of Pi Distribution in Barley Seedlings. Bio-protocol 4(13): e1175. DOI: 10.21769/BioProtoc.1175.
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Category
Plant Science > Plant physiology > Nutrition
Plant Science > Plant physiology > Ion analysis
Biochemistry > Other compound > Ion
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1,176 | https://bio-protocol.org/exchange/protocoldetail?id=1176&type=0 | # Bio-Protocol Content
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Peer-reviewed
Open-book Preparations from Chick Embryos and DiI Labeling of Commissural Axons
Nicole H. Wilson
Esther T. Stoeckli
Published: Vol 4, Iss 13, Jul 5, 2014
DOI: 10.21769/BioProtoc.1176 Views: 12348
Edited by: Xuecai Ge
Original Research Article:
The authors used this protocol in Aug 2013
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Aug 2013
Abstract
Successful neural circuit formation relies on the accurate navigation of axons towards their targets during development. Axons are guided by a combination of short-range and long-range, attractive and repulsive cues. The commissural axons of the developing spinal cord have provided an informative in vivo model for the identification of multiple axon guidance molecules and mechanisms. These axons extend ventrally from the dorsal spinal cord and cross the midline at the floor plate, before making a sharp rostral turn towards the head. This simple trajectory has facilitated the identification of many axon guidance molecules, because perturbation of the stereotypical guidance decisions as a result of genetic manipulations can be easily identified. The open-book assay is a method to assess the trajectory of spinal commissural axons. The spinal cord is dissected out, opened at the roof plate and pinned flat. Punctate injections of the lipophilic fluorescent dye, DiI, are used to trace commissural axon trajectories prior to microscopy and analysis.
Keywords: Axon guidance Spinal cord Neural development Dissection Axon tracing
Materials and Reagents
Fertilized eggs containing chicken embryos (incubated until Hamburger and Hamilton stage 25-26)
PBS (add 50 ml of 20x PBS to 950 ml of ddH2O)
Sylgard silicone elastomer (World Precision Instruments, catalog number: SYLG184 )
Fast DiI (Life Technologies, Molecular Probes®, catalog number: D-7756 ) (dissolved at 5 mg/ml in ethanol)
Vacuum grease (Dow Corning Corporation, catalog number: 976V )
20x sterile phosphate buffered saline (PBS) (see Recipes)
4% paraformaldehyde (4% PFA) (see Recipes)
Phosphate (PB) buffer (1 M, 1 L, pH 7.4) (see Recipes)
Equipment
Incubator set at 38.5 °C and 45% humidity (e.g. FIEM, Juppiter 576 Setter+Hatcher; Kendro Laboratory Products, Heraeus/Kendro, model: B12 )
Spring scissors (Fine Science Tools, catalog number: 15003-08 )
Dumont #5 forceps (Fine Science Tools, catalog number: 11252-20 )
90 mm Petri dishes (e.g. Thermo Fisher Scientific, Sterilin®, catalog number: 101VR20/C )
0.20 mm insect pins (Fine Science Tools, catalog number: 26002-20 )
0.10 mm insect pins (Fine Science Tools, catalog number: 26002-10 )
Microscope with adjustable transmitted light source (Leica MZ6 stereomicroscope with Leica CLS 150x )
Fine surgical scalpel (Grieshaber Logistic Group, catalog number: 68101 )
Tungsten wire (0.075 mm) (World Precision Instruments, catalog number: TGW0325 )
Metal flat spatula/spoon (e.g. Sigma-Aldrich, catalog number: S4022 , S3397 )
Borosilicate glass capillaries (outer Ø/inner Ø: 1.2 mm/0.68 mm) (World Precision Instruments, catalog number: 1B120F-4 )
Glass needle puller (Narishige Group, catalog number: PC-10 )
Polyethylene tubing (Ø 1.24 mm) (e.g. Angst and Pfister AG, catalog number: FT0110350018 )
Plastic transfer pipettes (e.g. Sarstedt, catalog number: 86.1171 )
18 G x 1.5” needle (e.g. Braun, catalog number: 4665120 )
1 ml syringe (e.g. Braun, catalog number: 9166017V )
20 ml syringe (e.g. Braun, catalog number : 4606205V )
24 mm x 24 mm glass coverslips (VWR International, catalog number: 631-0127 )
Microscope suitable for brightfield and fluorescent microscopy (e.g. Olympus, model: BX51 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wilson, N. H. and Stoeckli, E. T. (2014). Open-book Preparations from Chick Embryos and DiI Labeling of Commissural Axons. Bio-protocol 4(13): e1176. DOI: 10.21769/BioProtoc.1176.
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Category
Neuroscience > Development > Neuron
Neuroscience > Neuroanatomy and circuitry > Animal model
Developmental Biology > Cell growth and fate > Neuron
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1,177 | https://bio-protocol.org/exchange/protocoldetail?id=1177&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro EBV Infection of Mononuclear Cells that Have Been Cryo-preserved
Shanie Saghafian-Hedengren
ES Ebba Sohlberg
JT Jakob Theorell
CC Claudia Carvalho-Queiroz
NN Noémi Nagy
JP Jan-Olov Persson
CN Caroline Nilsson
YB Yenan T. Bryceson
ES Eva Sverremark-Ekström
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1177 Views: 7498
Original Research Article:
The authors used this protocol in Dec 2013
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Dec 2013
Abstract
Epstein-Barr Virus (EBV) is a B-lymphotropic herpesvirus which the majority of adult human population is latently-infected with. Various immunological and molecular in vitro studies have been facilitated by the use of EBV’s ability to infect and transform B cells to immortalized polyclonal B cell lines. Many of these studies use freshly isolated cord-blood mononuclear cells (CBMC). Some experiments may, however, require EBV infection of samples that have been prospectively collected and cryo-preserved. Here we share a protocol that we used to successfully infect B cells from cryo-preserved CBMCs and peripheral-blood mononuclear cells (PBMC) (Sohlberg et al., 2013; Saghafian-Hedengren et al., 2013).
Materials and Reagents
Cryo-preserved CBMC
Cryo-preserved PBMC
EBV strain B95-8 containing supernatant
Note: The batch here had a titer of 2.5 x 105 Ramos infectious units. RaIU, which was determined by infection of the EBV negative Burkitts lymphoma B-cell line Ramos, followed by anti-complement immunoflourescent assay (ACIF) to detect the number of infected cells.
RPMI 1640 (Life Technologies)
10% heat-inactivated fetal-calf serum (Hyclone)
L-glutamine (2 mmol/l)
Penicillin G-sodium (100 U/ml)
Streptomycin sulfate (100 mg/ml) (Merck KGaA)
Complete cell-culture medium (see Recipes)
Equipment
48-well flat-bottomed tissue-culture treated plates (Sarstedt AG)
Humified incubator with for adjustment of 37 °C and 5% CO2 for cell culture
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Saghafian-Hedengren, S., Sohlberg, E., Theorell, J., Carvalho-Queiroz, C., Nagy, N., Persson, J., Nilsson, C., Bryceson, Y. T. and Sverremark-Ekström, E. (2014). In vitro EBV Infection of Mononuclear Cells that Have Been Cryo-preserved. Bio-protocol 4(14): e1177. DOI: 10.21769/BioProtoc.1177.
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Category
Microbiology > Microbe-host interactions > Virus
Microbiology > Microbial cell biology > Cell isolation and culture
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1,178 | https://bio-protocol.org/exchange/protocoldetail?id=1178&type=0 | # Bio-Protocol Content
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Coculture between hMADS and Mouse Adult CM
Florence Figeac
Adrien Acquistapace
Olivier Le Coz
PL Pierre François Lesault
Anne-Marie Rodriguez
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1178 Views: 9706
Original Research Article:
The authors used this protocol in Jan 2014
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Jan 2014
Abstract
Heart failure occurring after acute myocardial infarction (MI) is among the main causes of death in western countries. Cell therapies, particularly those based on mesenchymal stem cells (MSC), represent one of the most promising approaches to repair damaged heart tissues. Several reports have provided evidences that injection of mesenchymal stem cells improved heart function following myocardial infarction (Shake et al., 2002; Zimmet and Hare, 2005; Zeng et al., 2007). Nevertheless, the mechanism(s) by which MSC exert their therapeutic action is far from being understood, and further knowledges in this field are required especially to optimize efficiency of current cardiac cell therapies. To assess the regenerative mechanisms developed by MSC in vitro, we developed the method described above which is expected to mimic the micro-environment typical of an infarcted heart. This method consists in a species mismatch coculture between mouse terminally differentiated cardiomyocytes in a distressed state and human Multipotent Adipose Derived Stem cells (hMADS cells) used herein as an MSC model.
Materials and Reagents
hMADS cells were isolated as described previously (Rodriguez et al., 2005)
Perfusion Buffer stock solution (sPB)
Liberase blendzyme TM (Roche Diagnostics, catalog number: 05401119001 )
Trypsin (Sigma-Aldrich, catalog number: T4549 )
Calcium chloride
Annexin V/7AAD staining (BD Biosciences, catalog number: 559763 )
18α-glycyrrhetinic acid (18α-GA) (Sigma-Aldrich, catalog number: G-8503 )
M Latrunculin A (Life Technologies, catalog number: L12370 )
Nocodazole (Sigma-Aldrich, catalog number: M-1404 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876 )
Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: M9397 )
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: P9144 )
1 M HEPES buffer solution (Sigma-Aldrich, catalog number: M3375 )
Taurine (Sigma-Aldrich, catalog number: T0625 )
Phenol Red (last) (Sigma-Aldrich, catalog number: P5530 )
5.5 mM glucose (Sigma-Aldrich, catalog number: G7528 )
0.25 mg/ml liberase blendzyme (Roche Diagnostics, catalog number: 1988417 )
Trypsin (Sigma-Aldrich, catalog number: T4549)
10% newborn calf serum (Life Technologies, Gibco®, catalog number: 16010167 )
5% bovine calf serum (BCS) (HyClone, catalog number: SH30073 )
Pentobarbital sodium (CEVA, CIP number: 6742145 )
Heparin choay (sanofi-aventis, CIP number: 3048450 )
1x perfusion buffer stock solution (see Recipes)
2,3-Butanedione monoxime (BDM) (Sigma-Aldrich, catalog number: B0753 ) (see Recipes)
Perfusion buffer (pH 7.46) (PB) (see Recipes)
Digestion buffer (see Recipes)
Stopping buffer 1 (see Recipes)
Stopping buffer 2 (see Recipes)
Equipment
Langendorff apparatus (see below)
Figure 1. Langendorff apparatus
Water bath at 37 °C
Surgical silk 4.0
Petri dishes (Dutscher Scientific, catalog number: 628103 )
Surgical instruments
Disposable transfer pipets (VWR International, catalog number: 612-1681 )
Falcon 15 ml propylene conical tubes (Dutscher Scientific, catalog number: 352095 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Stem Cell > Adult stem cell > Mesenchymal stem cell
Stem Cell > Adult stem cell > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell differentiation
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1,179 | https://bio-protocol.org/exchange/protocoldetail?id=1179&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation of ILC2 from Mouse Liver
Tamar Mchedlidze
Stefan Wirtz
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1179 Views: 12375
Reviewed by: Savita Nair
Original Research Article:
The authors used this protocol in Aug 2013
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The authors used this protocol in:
Aug 2013
Abstract
Group 2 innate lymphoid cells (ILC2) are a recently characterized cell population which lacks specific antigen receptors and contributes to immune responses at mucosal surfaces of lung and gut. Recently, we demonstrated that ILC2 expand in the context of chronic liver diseases in mice and contribute to pathology in an IL-33 dependent manner. Here, we describe a protocol to isolate highly purified ILC2 from mouse livers. This procedure provides effective digestion of liver tissue and limits proteolytic degradation of cell surface receptors leading to increased yields of biologically functional cells. The number of liver resident ILC2 in the steady state is low, however their number dramatically increase upon systemic or local treatment of mice with cytokines such as IL-25 and IL-33. Using minicircle-based expression constructs for these cytokines high numbers of functional ILC2 can be isolated with the protocol provided here.
Keywords: Group 2 innate lymphoid cells ILC2 Inflammation
Materials and Reagents
Liver from a IL-33 or IL-25 treated mouse (e.g. C57BL/6 or Balb/c)
Fetal calf serum (FCS)
RPMI-1640 medium
Easycoll separation solution (density 1.124) (Biochrom, catalog number: L6145 )
Anti-mouse CD16/CD32 (Fc-Block) (eBioscience, catalog number: 14-0161 )
0.5 M CaCl2 solution
0.2 M MgCl2 solution
AutoMACS rinsing solution (Miltenyi Biotec)
Brilliant Violet 421 Streptavidin (BioLegend, catalog number: 405226 )
Antibodies
FACS antibodies
Anti-Sca-1 (clone D7, FITC conjugated) (eBioscience, catalog number: 11-5981 )
Anti-KLRG1 (clone 2F1, APC conjugated) (eBioscience, catalog number: 17-5893 )
Anti-ICOS (clone 7E.17G9, PE conjugated) (eBioscience, catalog number: 12-9942 )
Anti-mouse lineage-antibodies biotin labelled
Anti-CD3 (clone 145-2C11) (eBioscience, catalog number: 13-0031 )
Anti-CD45R (clone RA3-6B2) (eBioscience, catalog number: 13-0452 )
Anti-Ly-6G (clone RB6-8C5) (eBioscience, catalog number: 13-5931 )
Anti-CD11b (clone M1/70) (eBioscience, catalog number: 13-0112 )
Anti-NK1.1 (clone PK136) (eBioscience, catalog number: 13-5941 )
Anti-Ter-119 (clone Ter-119) (eBioscience, catalog number: 13-5921 )
Anti-SiglecF (clone ES22-10D8) (Miltenyi Biotec, catalog number: 130-101-861 )
Anti-CD5 (clone 53-7.3) (Miltenyi Biotec, catalog number: 130-101-960 )
Krebs-Ringer-Buffer (KRB) (see Recipes)
Collagenase IV solution (Sigma-Aldrich, catalog number: C5138 ) (see Recipes)
DNase I solution (Roche Diagnostics, catalog number: 10104159001 ) (see Recipes)
30% Biocoll (see Recipes)
80% Biocoll (see Recipes)
FACS buffer (see Recipes)
ACK buffer (see Recipes)
PEB buffer (see Recipes)
Equipment
GentleMACS C tube (Miltenyi Biotec)
6 well plates
GentleMACS dissociator (Miltenyi Biotec)
MACSmix tube rotator (Miltenyi Biotec)
Centrifuge (Thermo Fisher Scientific, model: Multifuge X1R )
Cell sorter (e.g. FACSaria, BD Biosciences)
100 µm cell strainer (Corning, catalog numer: 08-771-19 )
Hemocytometer
15 ml Falcon tubes
50 ml Falcon tubes
FACS tubes
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Mchedlidze, T. and Wirtz, S. (2014). Isolation of ILC2 from Mouse Liver. Bio-protocol 4(14): e1179. DOI: 10.21769/BioProtoc.1179.
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Category
Immunology > Immune cell isolation > Lymphocyte
Cell Biology > Cell isolation and culture > Cell isolation
Cell Biology > Tissue analysis > Tissue isolation
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118 | https://bio-protocol.org/exchange/protocoldetail?id=118&type=1 | # Bio-Protocol Content
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Peer-reviewed
Alarmablue Assay for Detecting Cell Viability
HZ Huagang Zhang
Published: Aug 20, 2011
DOI: 10.21769/BioProtoc.118 Views: 14683
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Abstract
AlamarBlueTM detects cell viability by utilizing a nonfluorescent dye resazurin, which is converted to a fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth. The fluorescent or colorimetric signal generated from the assay is proportional to the number of living cells in the sample (detailed information can be found from here).
Materials and Reagents
Resazurin cell viability assay kit (Biotium, catalog number: 30025 )
Cells: in this protocol, three human prostate cancer cell lines are tested.
DU 145 (ATCC, catalog number: HTB-81 ™)
PC3 (ATCC, catalog number: CRL-1435 ™)
LNCap (ATCC, catalog number: CRL-1740 ™)
Equipment
Spectra PLUS microplate reader
96-well tissue culture plates
Procedure
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhang, H. (2011). Alarmablue Assay for Detecting Cell Viability. Bio-101: e118. DOI: 10.21769/BioProtoc.118.
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Category
Cancer Biology > General technique > Cell biology assays > Cell viability
Cell Biology > Cell staining > Whole cell
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1,180 | https://bio-protocol.org/exchange/protocoldetail?id=1180&type=0 | # Bio-Protocol Content
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Macrophage Inflammatory Assay
Thomas J. Bartosh
Joni H. Ylostalo
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1180 Views: 30236
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The authors used this protocol in Nov 2013
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Abstract
Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al., 2010; Ylostalo et al., 2012; Bartosh et al., 2013). The assay can be adapted appropriately to test macrophage response to other agents as well that will be referred to herein as ‘test reagents’ or ‘test compounds’.
In this protocol, the mouse macrophage cell line J774A.1 is expanded as an adherent monolayer on petri dishes allowing for the cells to be harvested easily without enzymes or cell scrapers that can damage the cells. The macropahges are then stimulated in suspension with LPS and seeded into 12-well cell culture plates containing the test reagents. After 16-18 h, the medium conditioned by the macrophages is harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA). We routinely measure levels of the pro-inflammtory cytokine TNF-alpha and the anti-inflammatory cytokine interleukin-10 (IL-10).
Keywords: Macrophage LPS Inflammation Prostaglandin E2 IL10
Materials and Reagents
J774A.1 mouse macrophages (ATCC, catalog number: TIB-67 )
0.1 mg/ml lipopolysaccharide (LPS) (Sigma-Aldrich, catalog number: L4130 ) solution in PBS
Mouse TNF-alpha Quantikine ELISA kit (R&D Systems, catalog number: MTA00B )
Mouse Interleukin 10 (IL-10) Quantikine ELISA kit (R&D Systems, catalog number: M1000B )
High glucose Dulbecco’s modified Eagle medium (DMEM) containing Glutamax (Life Technologies, catalog number: 10569 )
Fetal bovine serum (Atlanta Biologicals, catalog number: S11550 )
Penicillin-streptomycin (Life Technologies, catalog number: 15140 )
Macrophage medium (see Recipes)
Equipment
150 x 15 mm petri dish (BD Biosciences, Falcon®, catalog number: 351058 )
Stericup-GP 0.22 µm vacuum filtration device (EMD Millipore, catalog number: SCGPU05RE )
12-well tissue culture treated plates (Corning, catalog number: 3512 )
10 ml capacity serological pipette (VWR International, catalog number: 89130 )
1.5 ml microcentrifuge tubes
50 ml sterile conical tube (BD Biosciences, Falcon®, catalog number: 352070 )
Water bath set to 37 °C
Pipette-aid
Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
Humidified cell culture incubator set to 37 °C and 5% CO2
Upright microscope with 10x objective
Microplate reader (capable of measuring absorbance at 450 nm, with a background correction wavelength of 540 nm or 570 nm)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bartosh, T. J. and Ylostalo, J. H. (2014). Macrophage Inflammatory Assay. Bio-protocol 4(14): e1180. DOI: 10.21769/BioProtoc.1180.
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Category
Immunology > Immune cell function > Macrophage
Immunology > Immune cell function > Cytokine
Cell Biology > Cell isolation and culture > Cell growth
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1,181 | https://bio-protocol.org/exchange/protocoldetail?id=1181&type=0 | # Bio-Protocol Content
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Mesenchymal Stem Cell (MSC) Aggregate Formation in vivo
Thomas J. Bartosh
Joni H. Ylostalo
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1181 Views: 11587
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The authors used this protocol in Nov 2013
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Abstract
Human mesenchymal stem/progenitor cells (MSCs) isolated from various adult tissues show remarkable therapeutic potential and are being employed in clinical trials for the treatment of numerous diseases (Prockop et al., 2010). While routes of cell administration vary, profound beneficial effects of MSCs in animal models have been observed following intraperitoneal injections of the cells (Roddy et al., 2011). Similar to MSC spheres formed in culture under conditions where attachment to plastic is not permitted (Bartosh et al., 2010), MSCs injected into the peritoneum of mice spontaneously aggregate into 3D sphere-like structures (Bartosh et al., 2013). During the process of sphere assembly and compaction, MSCs upregulate expression of numerous therapeutic anti-inflammatory and immune modulatory factors. Here we describe the method we previously used for the generation of human bone marrow-derived MSC aggregates/spheres in vivo (Bartosh et al., 2013). By tagging the MSCs with green fluorescent protein (GFP), the aggregates formed can be easily visualized, collected and analyzed for changes in cellular properties and interactions with host immune cells.
Keywords: MSCs Spheroids Immunomodulatory Peritoneum Antiinflammatory
Materials and Reagents
Human bone marrow mesenchymal stem cells expressing green fluorescent protein (GFP-MSCs) from The Center for the Preparation and Distribution of Adult Stem Cells (http://medicine.tamhsc.edu/irm/msc-distribution.html)
C57BL/6J or BALB/C mice (2-3 months of age) (The Jackson Laboratory)
Phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (pH 7.4) (Life Technologies, catalog number: 10010 )
Hank’s Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (Lonza, catalog number: 04-315Q )
0.25% trypsin with 1x EDTA (Life Technologies, catalog number: 25200 )
Minimum Essential Medium alpha (Life Technologies, catalog number: 12561 )
Premium select fetal bovine serum (Atlanta Biologicals, catalog number: S11550 )
Penicillin-streptomycin (Life Technologies, catalog number: 15140 )
100x L-glutamine (Life Technologies, catalog number: 25030 )
Complete culture medium (CCM) for MSC growth (see Recipes)
Equipment
Stericup-GP 0.22 µm vacuum filtration device (EMD Millipore, catalog number: SCGPU05RE )
Water bath set to 37 °C
Centrifuge with swinging-bucket rotor and adaptors for 50 ml conical tubes
50 ml sterile conical tube (BD Biosciences, Falcon®, catalog number: 352070 )
Humidified cell culture incubator set to 37 °C and 5% CO2
Upright microscope with 4x and 10x objectives and a filter set to visualize GFP
29 gauge needle with 1 ml syringe (Terumo Europe N.V., catalog number: 05M2913 )
Isoflurane anesthesia system with nose cone for mouse
Sterile dissecting scissors, pins, and curved forceps with a serrated edge
Rubber or styrofoam platform
Dissecting microscope with optional camera and monitor (Figure 1)
Illumatool Bright Lights Systems LT 9900 with epi-fluorescence attachment (Lightools Research) and GFP filter set (Figure 1)
Figure 1. Equipment required to visualize and collect GFP-MSC aggregates/spheres from the mouse peritoneum. GFP-MSC aggregates/spheres can be visualized in the mouse peritoneum using a dissecting microscope with an epi-fluorescence attachment and GFP filter set. High quality images can be acquired with an appropriate camera mounted to the dissecting scope (a camera is not a requirement for collecting the aggregates).
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bartosh, T. J. and Ylostalo, J. H. (2014). Mesenchymal Stem Cell (MSC) Aggregate Formation in vivo. Bio-protocol 4(14): e1181. DOI: 10.21769/BioProtoc.1181.
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Category
Stem Cell > Adult stem cell > Mesenchymal stem cell
Stem Cell > Adult stem cell > Maintenance and differentiation
Cell Biology > Cell isolation and culture > 3D cell culture
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1,182 | https://bio-protocol.org/exchange/protocoldetail?id=1182&type=0 | # Bio-Protocol Content
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Hedgehog (Hh) Reporter Activity Assay
Chen Zhao
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1182 Views: 14860
Edited by: Fanglian He
Reviewed by: Hong Lok Lung
Original Research Article:
The authors used this protocol in Jul 2010
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Abstract
This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.
Materials and Reagents
NIH3T3 cells or NIH3T3-Light2 cells (ATCC, catalog number: JHU-68 )
Note: NIH3T3-Light2 cells were initially generated in Dr. Beachy’s Laboratory and were deposited in ATCC, the public available source of biomedical reagents including laboratory made cell lines. This cell line is available from Dr. Beachy’s lab per request.
Hh-conditioned medium
Fetal bovine serum (FBS)
Bovine calf serum (BCS)
DMEM (high glucose) (Life Technologies, InvitrogenTM, catalog number: 11965 )
TransIT-2020 (Mirius, catalog number: MIR5404 )
8XGli-Firefly luciferase expression construct
Note: The 8XGli-Firefly luciferase expression construct is made at Dr. Beachy’s Laboratory. Eight consecutive repeats of Gli responding element (8XGli) are PCR amplified and cloned into the minimal enhancer region of pGL3-Luciferase [promega] vector. Map of pGL3 is available from promega.
pRL-SV40-Renilla luciferase expression construct
Note: The pRL-SV40-Renilla luciferase expression construct is made at Dr. Beachy’s Laboratory.
Dual-Luciferase @ Reporter Assay System (Promega Corporation, catalog number: E1960 )
Growth media (see Recipes)
Shh-conditioned media (see Recipes)
Serum deprived media (see Recipes)
Equipment
15 ml thermal scientific Nunc conical tubes
T75 culture flask (BD Biosciences, Falcon®, catalog number: 353135 )
24-well tissue culture plate (BD Biosciences, Falcon®, catalog number: 353047 )
96-well flat-bottom plate (Corning, Costar® , catalog number: 3915 )
Centrifuge (Eppendorf, model: 8810R )
Water bath
CO2 incubator
Berthold Luminometer (Berthold Technologies, model: Centro XS LB960 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhao, C. (2014). Hedgehog (Hh) Reporter Activity Assay. Bio-protocol 4(14): e1182. DOI: 10.21769/BioProtoc.1182.
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Category
Developmental Biology > Cell signaling > Ligand
Stem Cell > Embryonic stem cell > Maintenance and differentiation
Cell Biology > Cell signaling > Development
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1,183 | https://bio-protocol.org/exchange/protocoldetail?id=1183&type=0 | # Bio-Protocol Content
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Wnt Reporter Activity Assay
Chen Zhao
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1183 Views: 35221
Edited by: Fanglian He
Reviewed by: Hong Lok Lung
Original Research Article:
The authors used this protocol in Jul 2010
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Jul 2010
Abstract
This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.
Materials and Reagents
NIH3T3 cells or HEK293 cells
Purified active Wnt3a proteins (ATCC, catalog number: CRL2647 TM)
Fetal bovine serum (FBS)
DMEM (high glucose) (Life Technologies, InvitrogenTM, catalog number: 11965 )
TransIT-2020 (Mirus Bio LLC, catalog number: MIR5404 )
TCL/LEF-Firefly luciferase expression construct (Addgene, catalog number: 12456 )
pRL-SV40-Renilla luciferase expression construct
Note: The pRL-SV40-Renilla luciferase expression construct is made at Dr. Beachy’s Laboratory.
Dual-Luciferase @ Reporter Assay System (Promega Corporation, catalog number: E1960 )
Growth media (see Recipes)
Wnt3a-conditioned media (see Recipes)
Serum-deprived media (see Recipes)
Equipment
T75 culture flask (BD Biosciences, Falcon®, catalog number: 353135 )
24-well tissue culture plate (BD Biosciences, Falcon®, catalog number: 353047 )
96-well flat-bottom plate (Corning, Costar®, catalog number: 3915 )
Centrifuge (Eppendorf, model: 8810R )
Water bath
CO2 incubator
Berthold Luminometer (Berthold Tecnologies, model: Centro XS LB960 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Developmental Biology > Cell signaling > Ligand
Stem Cell > Embryonic stem cell > Maintenance and differentiation
Cell Biology > Cell signaling > Development
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1,184 | https://bio-protocol.org/exchange/protocoldetail?id=1184&type=0 | # Bio-Protocol Content
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Peer-reviewed
Affinofile Assay for Identifying Macrophage-Tropic HIV-1
SJ Sarah B. Joseph
BL Benhur Lee
RS Ronald Swanstrom
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1184 Views: 8427
Original Research Article:
The authors used this protocol in Jul 2013
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Abstract
The ability to enter monocyte-derived macrophage (MDM) in vitro is commonly used to define macrophage-tropic HIV-1 despite the fact that viruses vary continuously in their ability to enter MDMs in vitro, and MDMs vary in their ability to support HIV-1 entry (Joseph et al., 2014; Peters et al., 2006). This makes it difficult to distinguish viruses that are adapted to replicating in macrophage from those that are adapted to replicating in T cells. We use the Affinofile cell line ( Johnston et al., 2009) to assay for macrophage tropism by capitalizing on the fact that macrophage-tropic HIV-1 has an enhanced ability to enter cells expressing low levels of CD4 (Joseph et al., 2014; Peters et al., 2006; Duenas-Decamp et al., 2009; Dunfee et al., 2006; Gorry et al., 2002; Martin-Garcia et al., 2006; Peters et al., 2004) and Affinofile cells can be induced to express a wide range of CD4 densities (Johnston et al., 2009). We induce Affinofile cells to express either high or low CD4, infect those cells with pseudotyped reporter virus, and quantify percent infectivity at low CD4 relative to infectivity at high CD4. Macrophage-tropic viruses have an enhanced ability to infect at low CD4. Using this approach we have found that macrophage-tropic strains of HIV-1 are relatively rare and that most HIV-1 variants require high levels of CD4 to enter cells, a phenotype we have referred to as R5 T cell-tropic.
Materials and Reagents
Affinofile cells (Johnston et al., 2009)
Luciferase Assay System (Promega Corporation, catalog number: E1501 )
5x Reporter Lysis Buffer (Promega Corporation, catalog number: E397A )
PE-conjugated anti-CD4 antibody (clone RPA-T4) (BD, catalog number: 555347 )
PE-conjugated anti-CCR5 antibody (clone 2D7) (BD, catalog number: 555993 )
Aqua live/dead stain (Life Technologies, catalog number: L34957 )
Env-pseudotyped luciferase reporter virus stock (frozen and tittered)
Blasticidin (Life Technologies, catalog number: A11139-03 )
FBS (Atlanta Biologicals, catalog number: S12850 )
DMEM-H (Cellgro, catalog number: 10-013-CV )
10% Buffered Formalin (Thermo Fisher Scientific, catalog number: SF100 )
Poly-L-Lysine (Sigma-Aldrich, catalog number: P4707 ) (see Recipes)
Ponasterone A (Life Technologies, catalog number: 45-0478 ) (see Recipes)
Doxycycline (Sigma-Aldrich, catalog number: D9891 ) (see Recipes)
DMEM-F10/B (see Recipes)
1x staining solution (see Recipes)
Fixing solution (see Recipes)
Equipment
96-well plate (sterile, black) (Corning, Costar®, catalog number: 3916 )
24 well plate
37 °C, 5% CO2 cell culture incubator (BSL-2)
Hemocytometer
Light microscope
PE-conjugated QuantiBRITE beads (BD, catalog number: 340495 )
Incubated plate centrifuge (BSL-2)
Laminar flow biosafety cabinet (BSL-2)
Plate-reading luminometer
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Joseph, S. B., Lee, B. and Swanstrom, R. (2014). Affinofile Assay for Identifying Macrophage-Tropic HIV-1. Bio-protocol 4(14): e1184. DOI: 10.21769/BioProtoc.1184.
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Category
Microbiology > Microbe-host interactions > Virus
Microbiology > Microbial cell biology > Cell viability
Immunology > Immune cell function > Macrophage
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1,185 | https://bio-protocol.org/exchange/protocoldetail?id=1185&type=0 | # Bio-Protocol Content
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Chromatin Fractionation Assay in Fission Yeast
Tatsuki Kunoh
TH Toshiyuki Habu
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1185 Views: 11669
Reviewed by: Kanika GeraBelen Sanz
Original Research Article:
The authors used this protocol in Jan 2014
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Abstract
The protein recruitment onto chromatin is a critical process for DNA metabolism, including DNA replication, DNA repair and DNA recombination. Especially DNA modification enzymes and checkpoint proteins are loaded onto DNA damage sites in a context-dependent manner. In our recent study (Kunoh and Habu, 2014), the chromatin association of Pcf1, a large subunit of Chromatin Assembly Factor-1 (CAF-1), was monitored after exposure of cells to hydroxyurea which slowed down the DNA replication. Results of the chromatin fractionation assay provided evidence that Pcf1 was recruited to chromatin upon DNA replication stress. A similar procedure enabled to reveal the chromatin association of Orp1, Mcm proteins, and Swi6 (Sadaie et al., 2008; Ogawa et al., 1999). This assay allows us to fractionate chromatin-bound and -unbound proteins from living cells. The following immunoblot of the respective fractions provides the information concerning the chromatin binding status of our target proteins.
Keywords: Chromatin Fractionation Yeast Schizosaccharomyces pombe Pcf1
Materials and Reagents
Yeast strain (Schizosaccharomyces pombe)
Flask (IWAKI PUMPS, catalog number: 4980FK500 )
Conical tube (BD Biosciences, Falcon®, catalog number: 2070 )
1.5 ml microcentrifuge tube (Eppendorf, catalog number: 022364111 )
Lysing enzymes from Trichoderma harzianum (Sigma-Aldrih, catalog number: L1412 )
Zymolyase 100T (Seikagaku Corporation, catalog number: 120493-1 )
Complete Mini (Roche Diagnostics, catalog number: 11836153001 )
Anti-GFP antibody (Roche Diagnostics, catalog number: 11814460001 )
Anti-histone H3 antibody (Millipore, Upstate Biotechnology, catalog number: 05-499 )
Anti-alpha-tubulin antibody (generously provided by Dr. A. Baines)
Note: Commercially available antibodies against alpha-tubulin (such as Abcam, catalog number: ab6161 ) can be used.
Goat HRP conjugated-anti-mouse antibody (Life Technologies, Biosource, catalog number: A10551 )
YES liquid medium (see Recipes)
STOP buffer (see Recipes)
PEMS buffer (see Recipes)
2x HBS buffer (see Recipes)
Lysis buffer (see Recipes)
2x Laemmli protein sample buffer (see Recipes)
Equipment
Air- (Tykyo Rikakikai, Eyela, model: FMC-1000 ) or water bath- (Taitec, model: MM-10 ) incubator shaker
Centrifuges equipped with 50 ml tubes (Tomy Digital Biology, model: AX-501 ) and 1.5 ml microtubes (Tomy Digital Biology, model: MX-107 )
Heat block (TAITEC, model: DTU-1BN) or water bath (Taitec, model: EXN-B )
Light microscope (Nikon Corporation, model: Eclipse E200 )
Electrophoresis apparatus (Bio-Rad Laboratories, catalog numbers: 165-8002JA and 164-5052 )
Transfer unit (Bio-Rad Laboratories, catalog numbers: 170-3930JA and 170-3935JA )
Procedure
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How to cite：Kunoh, T. and Habu, T. (2014). Chromatin Fractionation Assay in Fission Yeast. Bio-protocol 4(14): e1185. DOI: 10.21769/BioProtoc.1185.
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Category
Microbiology > Microbial genetics > DNA
Molecular Biology > DNA > DNA-protein interaction
Molecular Biology > DNA > DNA damage and repair
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1,186 | https://bio-protocol.org/exchange/protocoldetail?id=1186&type=0 | # Bio-Protocol Content
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Development of a Novel Assay for Synthesis and Hydrolysis of Sedoheptulose 1,7-bisphosphate (SBP) in vitro by Combinations of Purified Fructose 1,6-bisphosphate aldolases (FBA) Proteins and Fructose 1,6-bisphosphatases (FBPase) Proteins from Bacillus methanolicus MGA3
Jessica Stolzenberger
SL Steffen N. Lindner
MP Marcus Persicke
TB Trygve Brautaset
Volker F. Wendisch
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1186 Views: 9001
Original Research Article:
The authors used this protocol in Nov 2013
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Abstract
Bacillus methanolicus (B. methanolicus) is a Gram-positive, thermotolerant, and facultative methylotrophic bacterium that can use the one-carbon (C1) compound methanol as a source of carbon and energy (Schendel et al., 1990; Arfman et al., 1997; Arfman et al., 1992). B. methanolicus uses the Ribulose monosphosphate (RuMP) cycle for the fixation of formaldehyde (Anthony, 1986; Brautaset et al., 2007). In the RuMP cycle, Sedoheptulose 1,7-bisphoasphate (SBP) can be produced from erythrose 4-phosohate (E4-P) and dihydroxyacetone phosphate (DHAP) by sedoheptulose 1,7-bisphosphate aldolase (SBA) and dephosphorylated to yield sedoheptulose 7-phosphate (S7-P) by sedoheptulose 1,7-bisphosphatase (SBPase). Unfortunately, since neither E4-P nor SBP is commercially available, these compounds cannot be used directly in enzyme assays to obtain evidence for synthesis and hydrolysis of SBP. To circumvent this limitation, a coupled discontinuous enzyme assay including transketolase from Saccharomyces cerevisiae (S. cerevisiae) was used. E4-P and xylulose 5-phosphate (XU5-P) were generated from fructose 6-phosphate (F-6P) and glyceraldehyde 3-phosphate (GAP) by transketolase from S. cerevisiae. Aldol condensation of E-4P with DHAP to yield SBP was tested by using purified fructose 1,6-bisphosphate aldolase (FBAC or FBAP) from B. methanolicus (Stolzenberger et al., 2013a). Subsequently, hydrolysis of SBP to S7-P was assayed by using purified fructose 1,6-bisphosphatase (GlpXC or GlpXP) from B. methanolicus (Aldolases and phosphatases are not commercially available.) (Stolzenberger et al., 2013b).
Keywords: Sedoheptulose 1,7-bisphosphate aldolase Fructose 1,6-bisphosphate aldolase Methylotrophy RuMP cycle Ribulose monophosphate cycle
Materials and Reagents
Transketolase (TKT) from S. cerevisiae (Sigma-Aldrich)
Purified proteins: Fructose 1,6-bisphosphatase (GlpXC) and promiscuous fructose 1,6-bisphosphatase/Sedoheptulose 1,7-bisphosphatase (GlpXP), as well as promiscuous fructose 1,6-bisphosphate aldolase/ Sedoheptulose 1,7-bisphosphate aldolase (FBAC and FBAP) from B. methanolicus MGA3
Sephadex G25 gel filtration (Amersham Biosciences )
Factor Xa (Novagen)
*(1 M) 50 mM Tris-HCl (pH 7.5)
*(400 mM) 20 mM D-fructose 6-phosphate dipotassium salt (F6-P) (Sigma-Aldrich)
*(400 mM) 20 mM DL-glyceraldehyd 3-phosphate solution (GAP) (Sigma-Aldrich)
*(400 mM) 20 mM dihydroxyacetone phosphate dilithium salt (DHAP)
*(200 mM) 10 µM thiamine pyrophosphate (TPP) (Sigma-Aldrich)
*(40 mM) 2 mM MnCl2
*(200 mM) 10 mM ammonium bicarbonate solution (pH 9.3)
Note: *Concentration of stock solution are given in brackets.
Equipment
Amicon Ultra-0.5 centrifugal filter (Millipore)
LaChromUltra HPLC system (Hitachi)
MicroTOF-Q hybrid quadrupole/time-of-flight mass spectrometer (BD)
SeQuant ZIC-pHILLIC column (150 x 2.1 mm) (Merck KGaA)
Software
Compass software 1.3 (BD)
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Stolzenberger, J., Lindner, S. N., Persicke, M., Brautaset, T. and Wendisch, V. F. (2014). Development of a Novel Assay for Synthesis and Hydrolysis of Sedoheptulose 1,7-bisphosphate (SBP) in vitro by Combinations of Purified Fructose 1,6-bisphosphate aldolases (FBA) Proteins and Fructose 1,6-bisphosphatases (FBPase) Proteins from Bacillus methanolicus MGA3. Bio-protocol 4(14): e1186. DOI: 10.21769/BioProtoc.1186.
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Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Synthesis
Biochemistry > Protein > Activity
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1,187 | https://bio-protocol.org/exchange/protocoldetail?id=1187&type=0 | # Bio-Protocol Content
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Bone Resorption Assay
Carina Scholtysek
GK Gerhard Krönke
GS Georg Schett
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1187 Views: 16454
Original Research Article:
The authors used this protocol in May 2013
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Abstract
The Bone resorption assay provides an easy to use protocol for quantitatively measuring in vitro osteoclast-mediated bone resorption. Osteoclasts can be seeded onto the bone slices and formation of resorption pits can be quantified via toluidinblue staining (Scholtysek et al., 2013).
Materials and Reagents
Osteoclasts
Bone slices (IDS PLC, catalog number: DT-1BON1000-96 )
Isopropanol
Toluidine blue O (Sigma-Aldrich, catalog number: 198161 )
MilliQ water
96 well plates for cell culture (Greiner Bio-One GmbH, catalog numer: 650185 )
Equipment
OsteoMeasure System (http://www.osteometrics.com/product_info.htm)
Ultrasonic water bath
Procedure
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How to cite：Scholtysek, C., Krönke, G. and Schett, G. (2014). Bone Resorption Assay. Bio-protocol 4(14): e1187. DOI: 10.21769/BioProtoc.1187.
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Category
Cancer Biology > General technique > Cell biology assays
Cell Biology > Cell staining > Whole cell
Cell Biology > Tissue analysis > Tissue staining
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1,188 | https://bio-protocol.org/exchange/protocoldetail?id=1188&type=0 | # Bio-Protocol Content
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Fluorescence Microscopy for Cilia in Cultured Cells and Zebrafish Embryos
JC Jingli Cao
X Xueliang Zhu
Xiumin Yan
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1188 Views: 14309
Reviewed by: Michelle Goody
Original Research Article:
The authors used this protocol in Jun 2012
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Abstract
Cilia are microtubule-based hair-like projections found in organisms, ranging from protozoa to mammals. This protocol provides methods for immunofluorescence staining of cilia in cultured cells and zebrafish embryos.
Keywords: Fluorecence microcopy Cilia Zebrafish Cultured cells
Materials and Reagents
hTERT-RPE1 cell line (ATCC , catalog number: CRL-4000 )
Zebrafish (AB strain) (China Zebrafish Resource Center)
DMEM/F12 medium (Life Technologies, InvitrogenTM, catalog number: 11330-032 )
Fetal bovine serum (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
Hygromycin B (Life Technologies, InvitrogenTM, catalog number: 10687010 )
Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
Pure ethanol (Sinopharm Chemical Regent, catalog number: 10009218 )
Low-melting-point agarose (Sigma-Aldrich, catalog number: A9414 )
Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
Triton X-100 (AMRESCO, catalog number: 0694 )
Tween-20 (AMRESCO, catalog number: 0777 )
Mouse monoclonal anti-acetylated tubulin antibody (Sigma-Aldrich, catalog number: T-6793 )
Goat anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-546 (Life Technologies, catalog number: A11030 )
Donkey anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-488 (Life Technologies, catalog number: A21202 )
4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D8417 )
Dako mounting medium (Dako, catalog number: s3023 )
PBS (see Recipes)
PBT (see Recipes)
4% paraformaldehyde (PFA) fixation solution (see Recipes)
Embryo fixation buffer (see Recipes)
Embryo blocking buffer (see Recipes)
4', 6-diamidino-2-phenylindole (DAPI) stock solution (see Recipes)
Complete growth medium (see Recipes)
Serum free medium (see Recipes)
75% ethanol (see Recipes)
Equipment
Millex-GP Filter unit with 0.22 µm pore size (Millipore, catalog number: SLGP033RS )
Forceps (The duMONT Company, catalog number: 0203-5/15-PO )
10 cm Petri dishes (BD Biosciences, Falcon®, catalog number: 353003 )
12-well plates (Corning, catalog number: 3513 )
2 ml Eppendorf (EP) tubes (Axygen, catalog number: MCT-200-C )
Glass slides (Fan Yi, catalog number: 7105P )
18mm diameter circle cover slips (Thermo Fisher Scientific, catalog number: 12-545-84 )
Parafilm (Parafilm M®, catalog number: PM-996 )
100 ml beaker (Thermo Fisher Scientific, catalog number: 1201-0100 )
Filter paper (GE, catalog number: 10311611 )
Plastic transfer pipette (Thermo Fisher Scientific, catalog number: 11387873 )
Shakers (Qilinbeier, catalog number: TS-8S )
Leica TCS SP5 confocal microscope equipped with a 63x oil objective and a 63x NA1.2 water immersion objective
Procedure
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Category
Developmental Biology > Morphogenesis > Motility
Cell Biology > Cell imaging > Fluorescence
Cell Biology > Cell staining > Nucleic acid
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1,189 | https://bio-protocol.org/exchange/protocoldetail?id=1189&type=0 | # Bio-Protocol Content
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Rice Meiotic Chromosome Spread Preparation of Pollen Mother Cells
XL Xingwang Li
CW Changyin Wu
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1189 Views: 10833
Edited by: Ru Zhang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Oct 2013
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Abstract
In this protocol, we describe a simple and efficient method for meiotic chromosome spread preparation in rice pollen mother cells. Meiotic chromosome preparation by spreading itself is an important technique for plant cytogenetics (Higgins et al., 2004; Chelysheva et al., 2012; Wang et al., 2009); furthermore, it is a crucial step for applying other cytogenetic methods including Fluorescence in situ hybridization (FISH) and immunostaining.
Materials and Reagents
Young panicles containing meiocytes of rice (Oryza sativa ssp japonica cv. Zhonghua 11)
70%, 90% and 100% ethanol (Analytical Reagents)
60% (v/v) acetic acid (Analytical Reagents)
Carmine (Sigma-Aldrich, catalog number: C1022-25G )
Liquid nitrogen
Vectashield Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200 )
Carnoy’s fixative (see Recipes)
Aceto-carmine (see Recipes)
Equipment
Stereo microscope
Fluorescence microscope (Zeiss, model: AX10 )
Microscopic slides and cover slips
Dissection needles and fine forceps
Heating block
CCD camera (Hamamatsu Photonics K.K., model: ORCA-R2 C10600 )
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Li, X. and Wu, C. (2014). Rice Meiotic Chromosome Spread Preparation of Pollen Mother Cells. Bio-protocol 4(14): e1189. DOI: 10.21769/BioProtoc.1189.
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Category
Plant Science > Plant molecular biology > DNA
Plant Science > Plant cell biology > Cell imaging
Molecular Biology > DNA > DNA structure
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119 | https://bio-protocol.org/exchange/protocoldetail?id=119&type=0 | # Bio-Protocol Content
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Gene Networks Based on the Graphical Gaussian Model
Shisong Ma
Published: Vol 2, Iss 4, Feb 20, 2012
DOI: 10.21769/BioProtoc.119 Views: 14561
Original Research Article:
The authors used this protocol in Nov 2007
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Nov 2007
Abstract
This protocol describes how to build a gene network based on the graphical Gaussian model (GGM) from large scale microarray data. GGM uses partial correlation coefficient (pcor) to infer co-expression relationship between genes. Compared to the traditional Pearson’ correlation coefficient, partial correlation is a better measurement of direct dependency between genes. However, to calculate pcor requires a large number of observations (microarray slides) greatly exceeding the number of variables (genes). This protocol uses a regularized method to circumvent this obstacle, and is capable of building a network for ~20,000 genes from ~2,000 microarray slides. For more details, see Ma et al. (2007). For help regarding the script, please contact the author.
Data and Software
Data
Large-scale microarray data:
The microarray data should be derived from the same platform, preferably from Affymetrix slides. Some good examples are: Affymetrix Arabidopsis ATH1 Genome Array, Affymetrix Human Genome U133 Plus 2.0 Array, and Affymetrix Mouse Genome 430 2.0 Array. A recommended place to search for this type of data is at the gene expression omnibus from NCBI (http://www.ncbi.nlm.nih.gov/geo/). The number of slides should be larger than 1,000.
Software
R (http://www.r-project.org/)
The GeneNet package for R:
(http://www.uni-leipzig.de/~strimmer/lab/software/genenet/index.html)
Cytoscape (http://www.cytoscape.org/)
Perl and C++ software environment
Equipment
Personal computer: Intel Core2 E6420 processor (or similar processing capability)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Systems Biology > Interactome > Gene network
Molecular Biology > DNA > Microarray
Systems Biology > Transcriptomics > Microarray
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1,190 | https://bio-protocol.org/exchange/protocoldetail?id=1190&type=0 | # Bio-Protocol Content
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Dye Release Experiments with Dextran Loaded Vesicles
Marc-Antoine Sani
NO Neil M. O’Brien-Simpson
FS Frances Separovic
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1190 Views: 9644
Original Research Article:
The authors used this protocol in Aug 2013
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Abstract
Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted with a chromophore. In this assay, dextran linked to rhodamine or fluorescein are both encapsulated in lipid vesicles to allow quantifying the leakage of each dextran individually from a single sample. For instance, the size evaluation of the lipid pore formed by an antimicrobial peptide has been recently achieved using this protocol (Sani et al., 2013).
Keywords: Model membranes Phospholipid bilayers Dye leakage Fluorescence assay Antimicrobial peptides
Materials and Reagents
Rhodamine-dextran (RD) of different molecular weights [for instance 40 kDa RD molecular weight (RD-40)]
Fluorescein-dextran (FD) of different molecular weights [for instance 4.4 kDa FD (FD-4)]
Tris.HCl
NaCl
MQ-water
Triton-X100
Peptide of interest e.g. Maculatin 1.1 (Sani et al., 2013)
Lipids of interest e.g. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL) (Sani et al., 2013)
Equipment
Polycarbonate membrane filters (200 nm diameter)
Extruder (for instance Avanti Mini-extruder, Alabaster)
Bench centrifuge
Spectrofluorimeter and quartz cuvette
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Sani, M., O’Brien-Simpson, N. M. and Separovic, F. (2014). Dye Release Experiments with Dextran Loaded Vesicles. Bio-protocol 4(14): e1190. DOI: 10.21769/BioProtoc.1190.
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Category
Microbiology > Microbial biochemistry > Lipid
Biochemistry > Lipid > Lipid binding
Biochemistry > Protein > Interaction
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1,191 | https://bio-protocol.org/exchange/protocoldetail?id=1191&type=0 | # Bio-Protocol Content
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Bacterial Fluorescent-dextran Diffusion Assay
NO Neil M. O’Brien-Simpson
NP Namfon Pantarat
KW Katrina A. Walsh
ER Eric C. Reynolds
Marc-Antoine Sani
FS Frances Separovic
Published: Vol 4, Iss 14, Jul 20, 2014
DOI: 10.21769/BioProtoc.1191 Views: 8918
Original Research Article:
The authors used this protocol in Aug 2013
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Aug 2013
Abstract
Antimicrobial peptides are known to disrupt bacterial membranes allowing solutes to flow across the membrane in an unregulated manner resulting in death of the organism. Disrupting the bacterial membrane would thus perturb the cells osmotic balance resulting in an initial influx of the external aqueous buffer. We have designed an assay to investigate how antimicrobial peptide concentration affects the ability of fluorescently labelled dextran moieties of differing molecular weight and hydrodynamic radii to cross membranes of viable bacteria. This assay was used to show that diffusion of low and high molecular weight dextrans into bacteria was a function of antimicrobial peptide concentration (Sani et al., 2013).
Keywords: Bacterial membranes Antimicrobial peptides Fluorescent dye Cell osmotic balance Membrane diffusion
Materials and Reagents
Bacteria of interest [e.g. Staphylococcus aureus (S. aureus)] (Sani et al., 2013)
Rhodamine-dextran (RD) of different molecular weights [e.g. 40 kDa RD molecular weight (RD-40) (Sigma-Aldrich, catalog number: 42874 )]
Fluorescein-dextran (FD) of different molecular weights [e.g. 4.4 kDa FD (FD-4) (Sigma-Aldrich, catalog number: 46944 )]
LIVE/DEAD® BacLightTM Bacterial Viability and Counting Kit (Life Technologies, catalog number: L34856 )
Dulbecco’s Phosphate Buffered Saline (Dulbecco’s PBS) (Sigma-Aldrich, catalog number: D8537 )
Peptide of interest (e.g. Maculatin 1.1) (Sani et al., 2013)
Bacterial growth media [i.e., particular for the bacteria of interest e.g. S. aureus was grown as a batch culture in Luria broth (Thermo Fisher Scientific) (see Recipes)] (Sani et al., 2013)
Equipment
Flow cytometer (e.g. Beckman Coulter, model: Quanta SC-MPL)
96 well plates (flat bottomed) (Thermo Fisher Scientific, Nunc®, catalog number: 12565210 )
Transfer pipette
Procedure
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：O’Brien-Simpson, N. M., Pantarat, N., Walsh, K. A., Reynolds, E. C., Sani, M. and Separovic, F. (2014). Bacterial Fluorescent-dextran Diffusion Assay. Bio-protocol 4(14): e1191. DOI: 10.21769/BioProtoc.1191.
Download Citation in RIS Format
Category
Microbiology > Antimicrobial assay > Peptide assay
Microbiology > Microbial cell biology > Cell viability
Biochemistry > Carbohydrate > Polysaccharide
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