T Cell Activation and Cytokine Analysis

Fresh whole blood samples collected in heparin were diluted tenfold with RPMI and 90 µl of the diluted blood was incubated in 96-well flat-bottom tissue culture plates in a humidified incubator at 5% CO₂, 37ºC for 2 days in the presence or absence of anti-CD3 + anti-CD28. Specifically, to stimulate the T cells the plates were pre-coated overnight with 2 µg/mL of anti-human CD3 (clone OKT3, eBioscience, San Diego, CA). Anti-human CD28 (clone CD28.2, eBioscience, San Diego, CA) was then added at a final concentration of 1 µg/mL to each well. Alternatively, PBMCs isolated as for immunophenotyping above were stimulated anti-CD3 and anti-CD28 as previously described^{21 (link)}. PBMCs were counted using a Vi-Cell XR cell viability analyzer (Beckman Coulter, Brea, CA) and resuspended at 10⁶ cells/mL in RPMI + 10% autologous plasma + 100 U/mL penicillin/streptomycin. The PBMCs were aliquoted (50 μl/well) into 96-well flat-bottom tissue culture plates pre-coated or not as above with 0.5 µg/mL of anti-human CD3 and then 2 µg/mL anti-human CD28 added. After four days of incubation at 37 ˚C, 5% CO₂, the plates were then centrifuged at 300 × g in a Beckman TJ-6 centrifuge for 5 min and the supernatants collected for IFNγ analysis.