Activation of unprimed T cells with plate-bound anti-CD3/CD28 for T cell proliferation assay

 

  1. Dilute purified anti-CD3 (no azide / low endotoxin) to 10 ug/mL, 5 ug/mL, 2.5 ug/mL and 1.25 ug/mL, and purified anti-CD28 (NALE) to 10 ug/mL with sterile PBS.
  2. Coat a 96-well round-bottom plate with 50uL of each concentration of the anti-CD3/CD28 cocktail in triplicate. Include control wells of 50uL of sterile PBS only and media alone (not coated).
  3. Cover the plate and gently tap the side to ensure complete covering of the bottom of the wells. Seal plate with gladwrap or parafilm and incubate at 4°C overnight.
  4. Wash the wells gently with 200uL of sterile PBS twice.
  5. Resuspend 5 x 10^5 cells in 200uL of media in triplicate and add to each well. Include control wells of 200uL media alone.
  6. Cover the plate and incubate in a humidified chamber with 5% CO 2 at 37°C for 48 hours.
  7. Add 1 uCi (10uL of 1:10 dilution) of 3H-thymidine to each well and pulse cells in a humidified chamber with 5% CO2 at 37°C for 18 hours.
  8. Harvest cells onto a glass fibre filter and dry filter for at least 2 hours.
  9. Add liquid scintillant and measure 3H-thymidine incorporation with a beta-counter.
  10. Express results as mean cpm for triplicate cultures with standard deviation.