Shreffler:JAX Tcell studies

Overview

This is a protocol designed for characterizing the mouse allergen (Mus m1)-specific T cell phenotype during the JAX study.

Materials

• On average, 18mL of whole heparinized blood/patient will be given.
• target to isolate 8 x 10⁶ (8 million) cells for 48 hour culture
• Ficoll Paque Plus [Endotoxin tested], room temperature.
• Staining buffer (PBS + 5g BSA/L + 2mM EDTA)
• Sterile phosphate buffered saline (PBS), room temperature
• AIM V Media (w/HSA). Invitrogen # 0870112DK (Expires 1 month after opening)
• Cluster tubes
• Cryovial tubes
• Turk Blood Diluting Fluid (RICCA # 8850-16).
• Sterile conical tubes (15mL, 50mL)
• CD3, CD28 expander beads (Invitrogen 111-61D)
• Sterile, graduated transfer pipettes.
• Mus M1 (50μL) aliquot tube.
• Hemocytometer or disposable slides for the automated counter or manual counter
• 50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)
• Sterile 5mL polypropylene round-bottom tubes
• β-mercaptoethanol (Sigma #M7522)
• RLT lysis buffer (Qiagen, #79216)

Isolation of Mononuclear Cells

(Sterile Technique)

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. Place 15 mL of Ficoll in a 50 mL accuspin conical tube. Centrifuge at 800 g for 1 minute.
5. Dilute remaining blood at 1:1 with PBS in 50 mL conical tubes.
6. Add up to 30mL of diluted blood to the accuspin tube.
7. Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
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.)
9. Add PBS to bring to a minimum of 3x the volume, inverting up and down to mix.
10. Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).
11. Aspirate and discard the supernatant.
12. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding PBS for a total volume of 1 mL.
13. 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 100 μL of Turks Solution. Mix well, and let it sit for 1 minute before counting.
14. Add 19 mL of AIM-V to the cells in 990 μL to make a total volume of ~20 mL.
15. Centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).
16. 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.
17. Carefully introduce 10 μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter.
18. 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.
19. After centrifugation is completed, aspirate and discard the supernatant.
20. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with AIM-V at 4 x 10⁶/ml in 15 ml PP conical tube.

PBMC Stimulation

(Sterile Technique)

1. Remove Musm1 from the freezer and thaw.
2. Centrifuge the Musm1 briefly in the small centrifuge.
3. Label 5 mL sterile polypropylene tubes 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 (negative control): AIM-V medium alone.
   3. Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
4. Prepare solutions for each stimulant condition as follows:
   • 10 μg/mL MusM1 in AIM-V:
     • place 450 μL of AIM-V in the appropriately labeled tube
     • add 50 μL of allergen (MusM1)
     • add 500 μL of cell suspension
     • pipette up and down to mix
   • AIM-V medium alone:
     • place 500 μL of AIM-V in the appropriately labeled tube
     • add 500 μL of cell suspension
     • pipette up and down to mix
   • anti-CD3, anti-CD28 beads in AIM-V:
     • place 500 μL of AIM-V in the appropriately labeled tube
     • add 2.5 μL of CD3, CD28 expander beads
     • add 500 μL of cell suspension
     • pipette up and down to mix
   • Place the culture tubes with tube caps loose in the 37°C CO₂ incubator for 48 hours.

Collection of Supernatants

(non-sterile technique)

Note: Cell culture supernatants will be collected for cytokine measurement after 48 hour culture.

1. Visually inspect the cell cultures and make a note of variations in pellet size, culture media color, signs of contamination.
2. Mix the cells carefully by pipetting up and down to resuspend cells in the tube and evenly mix any secreted cytokine.
3. Take a 10 μL aliquot of each culture to inspect under the microscope.
4. Mix cell aliquot with equal volume of Turks solution and load 10 μL on a hemacytometer.
5. Note variations in cell density, obvious contamination.
6. Centrifuge tubes at 400g for 5 minutes at 25°C.
7. Obtain a cluster tube rack for the storage of supernatants.

   Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.

8. 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
9. For each stimulant, using a 1000μL pipette tip, transfer 380 μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack.

   Take care to not disturb the cell pellets.

10. Cap the cluster tubes and store in the –80°C freezer.

Collection of Cell Pellet for RNA Isolation

(non-sterile technique)

1. Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL of β-mercaptoethanol stock solution per 1ml of the RLT buffer.

   Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil

2. Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.
3. Centrifuge cells at 400 g for 5 minutes at RT. Please take special care to minimize disturbing the cell pellet.
4. Aspirate supernatant down to line at tube curvature (~50 μL ) using a pasteur pipette with unfiltered 200 μL pipette tip.
5. 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.
6. Vortex each tube twice for 15 seconds each time.
7. Transfer to labeled cryovial tube.
8. Freeze immediately and store for shipment.
9. Ship to MGH on dry ice.

Notes

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