unprimed T cells with plate-bound anti-CD3/CD28 for T cell proliferation
- 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.
- 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).
- 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.
- Wash the wells gently with 200uL of sterile PBS twice.
- Resuspend 5 x 10^5 cells in 200uL of media in triplicate and
add to each well. Include control wells of 200uL media alone.
- Cover the plate and incubate in a humidified chamber with 5%
CO 2 at 37°C for 48 hours.
- 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
- Harvest cells onto a glass fibre filter and dry filter for at
least 2 hours.
- Add liquid scintillant and measure 3H-thymidine incorporation
with a beta-counter.
- Express results as mean cpm for triplicate cultures with