Shreffler:JAX Tcell studies

Overview

This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.

Materials

• On average, 18mL of whole heparinized blood/patient will be given.
• 8 x 10⁶ (8 million) cells for 7 day culture (for measurement of frequency of PBMC-derived, Mus M1-specific CD4+ CD25+ FoxP3+ T cells)
• Sterile 5mL polystyrene round-bottom tubes.
• Phosphate buffered saline (PBS)

• Ficoll Paque Plus [Endotoxin tested], room temperature.
• CFSE (5μM aliquots).
• Staining buffer (PBS + 5g BSA/L + 2mM EDTA)

• Sterile phosphate buffered saline (PBS), room temperature
• AIM V Media (w/HSA). Invitrogen # 12055-091 add 5mL PS (PCN streptomycin), and 5mL Glutamine] (Expires 1 month after opening)
• Cluster tubes or Cryovial tubes and racks.

• 0.2% and 0.4% Trypan solution.
• IL-2 (approximately 20 units/μL; 1 μg = 2.4x10³ units) [To make a 10 μg vial of IL-2, reconstitute in 1mL sterile PBS + 1% human AB serum to get a final concentration of 10 μg/mL. Place in aliquots of 50 μL and freeze at -80°].

• mAb cocktail including: CD25-PCy5 (10uL), CD4-PC7 (5uL), CD3-APC7 (5uL), Aqua live/ dead (2μL), CD127-PE (1uL)

• Sterile conical tubes (15mL, 50mL)
• CD3, CD28 expander beads (Invitrogen 111-61D)

• 1X Facs Lysis Buffer

• 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
• Tetanus Toxoid (stock 2 mg/mL).

• Fix/Perm Concentrate

• 50 mL Accuspin tubes, radiation sterilized. Sigma #A-2055
• 24 well tissue culture plates.

• Fix/Perm Diluent

|Sterile 5mL polypropylene round-bottom tubes

• FoxP3-Alexa 647 Antibody.
• 15mL conical tubes.
• 5mL polystyrene tubes

Isolation of Mononuclear Cells

This is a sterile procedure and all steps should be performed in a hood.

1. Turn on the hood. Bring Ficoll and PBS to 20°C in the hood.
2. Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subject information, i.e. ID #, date collected, date received.
3. If performing Basophil Activation assay on this sample, set aside 3mL of whole blood.
4. Dilute remaining blood at 1:1 with PBS in 50 mL conical tubes.
5. Place 15 mL of Ficoll in a 50 mL conical tube. Overlay with up to 30mL of diluted blood, adding it very slowly to make sure that the blood doesn’t mix with the Ficoll layer.
6. Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
7. Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50 mL conical tube. (Avoid aspirating the Ficoll.) Add PBS to bring to a minimum of 2x the volume, inverting up and down to mix.
8. Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).
9. Aspirate and discard the supernatant. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding 1 mL of PBS. Set aside a 10 µL aliquot of cells for counting as follows: place the 10 µL of cells into 90 µL of PBS in a small sterile eppendorf tube, and add 100 µL of Trypan Blue (0.2%) into the eppendorf tube. Mix well, and let it sit for 1 minute before counting.
10. Add 19 mL of PBS 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).
11. In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.
12. 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.
13. After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with PBS at 10 x 10⁶/ml in 15 ml PP conical tube.

PBMC Stimulation

1. Remove stimulants from the freezer and thaw.
2. Label 24 well plate with specimen ID and date (this is for the 7 days cell culture). Label each well with the appropriate stimulant condition, ordered by priority (for cases where there are insufficient cells to test all stimulants).
   1. Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.
   2. AIM-V/IL2 (negative control): AIM-V + IL-2 medium alone.
   3. Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
   4. Tetanus: 200 μg/mL tetanus in AIM-V.
3. Make 5 mL of PBS + CFSE (2x solution): add 10 μL of stock CFSE (5 mM) into 5 mL of PBS.
4. Add an equal volume (1:1 dilution) of freshly prepared 10 μM CFSE (in PBS) to the tube of cells.
5. Incubate in 37°C water bath for 10 minutes.
6. After incubation, wash the CFSE stained cells in 10 mL of AIM-V @ 300g for 10 minutes at RT. Aspirate supernatant after centrifugation.
7. Resuspend CFSE stained cells in AIM-V medium @ 4 x 10⁶ cells/mL (cell suspension). For plating, each well should contain 2 x 10⁶ cells.
8. Prepare AIM-V + 2X IL-2 solution (AIM-V/IL2) by adding 10 μL of IL-2 to 5 mL of medium in a 15 mL conical tube. Vortex gently.
9. Prepare solutions for each stimulant condition as follows:
   • AIM-V/IL-2 medium alone: place 500 μL of AIM-V/IL-2 in the appropriately labeled tube and add 500 μL of cell suspension. Pipette up and down and plate.
   • 1 μg/mL anti-CD3, anti-CD28 beads in AIM-V/IL-2: 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 and plate.
   • 200 μg/mL MusM1 in AIM-V/IL-2: place 450 μL of AIM-V/IL-2 in the appropriately labeled tube and add 50 μL of allergen (MusM1). Then add 500 μL of cell suspension. Pipette up and down, and plate.
   • 200 μg/mL tetanus in AIM-V/IL-2: place 475 μL of AIM-V/IL-2 in the appropriately labeled tube and add 25 μL of tetanus. Then add 500 μL of cell suspension. Pipette up and down and plate.
   • Place the tissue culture plate in the 37°C CO₂ incubator for 7 days.

==Collection of Supernatants== (non-sterile)

1. Cell culture supernatants will be collected for cytokine measurement for 7 day cultures.
2. At day 7, collect cells carefully by pipetting up and down to resuspend cells in the well, and transfer the total volume in each well into separate 5ml polystyrene tubes. Rinse each well with 200μl of staining buffer and add to corresponding PS tubes. Tap gently to mix.
3. Centrifuge tubes at 300g for 5 minutes at 25°C temperature.
4. Obtain a cluster tube rack for the storage of supernatants.
5. Label cluster tubes as follows:
   1. Specimen ID
   2. Date
   3. Supernatants - 7 day
   4. Stimulant condition 1-4
      1. Stimulant 1 = MusM1
      2. Stimulant 2 = AIM-V
      3. Stimulant 3 = Beads
      4. Stimulant 4 = Tetanus Toxoid

   For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the PS 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 (by the freight elevators).

T regulatory cell surface staining

1. Prepare mAb cocktail [50 μL of cocktail/flow tube; volume of mAb needed per flow tube: CD25-PCy5 (10 μL), CD4-PC7 (5 μL), CD3-APC7 (5 μL), Aqua live/ dead (2 μL), CD127-PE (1 μL):

   add staining buffer to “cocktail tube” to reach a total volume of 50 μl per flow tube. Vortex. Store in fridge (light-sensitive) until ready for use]

2. Add 1 mL of staining buffer to each tube and vortex.
3. Wash cells at 300 g for 5 minutes at 4°C. Decant supernatant.
4. Add 1mL of staining buffer to each tube and vortex.
5. Wash cells at 300 g for 10 minutes at 4°C. Decant supernatant.
6. Add 45 μL of the cocktail to each tube.
7. Incubate @ 4°C for 20-30 minutes.
8. Add 3mL of staining buffer to each tube and vortex.
9. Wash at 300 g for 10 minutes at 4°C. Decant supernatant.
10. To fix/freeze the cells, pipette 500μl of 1X Facs Lysis Buffer into each tube and incubate @ 25°C in the dark for 15 minutes. After the incubation, pipette 500μl of the 20% DMSO + PBS solution into each tube.
11. Transfer the 1ml of cells + 1X Facs Lysis Buffer + 20% DMSO/PBS solution into an appropriately labeled cluster tubes, and freeze at -80°C for shipment to MGH.

Notes

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