Shreffler:JAX Tcell studies

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  • Phosphate buffered saline (PBS)
  • Ficoll Paque Plus [Endotoxin tested], room temperature.
  • Sterile phosphate buffered saline (PBS), room temperature
  • Cluster tubes or Cryovial tubes and racks.
  • Sterile conical tubes (15mL, 50mL)
  • CD3, CD28 expander beads (Invitrogen 111-61D)
  • Sterile, graduated transfer pipettes.
  • Mus M1 (50μL) aliquot tube.
  • A solution of 20% DMSO + PBS.
  • Hemocytometer or disposable slides for the automated counter or manual counter
  • Sterile 5mL polypropylene round-bottom tubes
  • β-mercaptoethanol (Sigma #M7522)
  • RLT lysis buffer (Qiagen, #79216)


Isolation of Mononuclear Cells

(Sterile Technique)


  8. Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50mL conical tube. (Avoid aspirating the Ficoll.) Add PBS to bring to a minimum of 2x the volume, inverting up and down to mix.
  10. Aspirate and discard the supernatant. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding PBS for a total volume of 1 mL. Set aside a 10 μL aliquot of cells for counting as follows: place the 10 μL of cells into 90 μL of PBS, and add 10 μL of Turk's Solution. Mix well, and let it sit for 1 minute before counting.
  11. Add 19 mL of AIM-V to the cells in 990 μL to make a total volume of ~20 mL and centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).
  13. Carefully introduce 10 μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter. Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 μL of the stained cells onto a disposable slide and count using the automated cell counter.

PBMC Stimulation

(Sterile Technique)

  1. Remove stimulants from the freezer and thaw.
  2. Label 5 mL sterile polypropylene tubes, each well with the appropriate stimulant condition, ordered by priority (for cases where there are insufficient cells to test all stimulants).
  3. Prepare solutions for each stimulant condition as follows:
    • 200 μg/mL MusM1 in AIM-V: place 450 μL of AIM-V in the appropriately labeled tube and add 50 μL of allergen (MusM1). Then add 500 μL of cell suspension. Pipette up and down.
    • AIM-V medium alone: place 500 μL of AIM-V in the appropriately labeled tube and add 500 μL of cell suspension. Pipette up and down.
    • 1 μg/mL anti-CD3, anti-CD28 beads in AIM-V: place 500 μL of AIM-V in the appropriately labeled tube and add 2.5 μL of CD3, CD28 expander beads. Then add 500 μL of cell suspension. Pipette up and down.
    • Place the culture tubes in the 37°C CO2 incubator for 48 hours.

Collection of Supernatants

(non-sterile technique)

  1. Cell culture supernatants will be collected for cytokine measurement after 48 hour culture.
  2. After 48 hours, mix the cells carefully by pipetting up and down to resuspend cells in the tube to evenly mix any secreted cytokine.
  3. Centrifuge tubes at 300g for 5 minutes at 25°C.
  4. Obtain a cluster tube rack for the storage of supernatants.
  5. Label two cluster tubes for each culture tube as follows:
    1. Specimen ID
    2. Date
    3. Supernatants - 48 hour
    4. Stimulant condition 1-3
      1. Stimulant 1 = MusM1
      2. Stimulant 2 = AIM-V
      3. Stimulant 3 = Beads
    For each stimulant, using a 1000μL pipette tip, transfer 400μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets.
  6. Cap the cluster tubes and store in the –80°C freezer.

Collection of Cell Pellet for RNA Isolation

(non-sterile technique)

  1. Add 1mL of staining buffer to the cell pellet of each culture tube, vortex on low.
  2. Centrifuge cells at 400 g for 5 minutes at 4°C. Please take special care to minimize the cell pellet disturbing or lost. Use a 1000 μL pipetman to aspirate most of the supernatant removing the last ~100 μL with a 200 μL pipetman. Make sure that only filtered pre-sterilized tips are being used from this step and later.
  3. Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL of ~14.3M stock solution of β-mercaptoethanol per 1ml of the RLT buffer
  4. Use a filtered pipette tip to add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully. Vortex each Eppendorf tube twice for 15 seconds each time and spin briefly at 1,000g for few seconds to collect all liquid at the bottom of the tube. Transfer to labeled cryovial tube.
  5. Freeze immediately and store for shipment.
  6. Ship to MGH on dry ice.



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