For optimal stimulation of the transfected cells we recommend coating 96-wells plates for stimulation [Nunc Immuno™ Plate C96 Maxi Sorp™, Cat. No. 430 341] with 50 µl of a solution of anti- CD3 antibody [OKt 3; eBioscience, Cat. No. 14-0037-82; final concentration 1 ng/µl] and anti-CD28 antibody [15E8; Research Diagnostics Inc., Cat. No. RD1-111650clb; final concentration 2 ng/µl] or with a solution of a control antibody [purified ms IgG(K); BD-Pharmingen Cat. No. 554 721; final concentration 3 ng/µl]. After incubation for 5 hours at 37°C/5% CO2 wells are washed three times with PBS containing 0.5% (w/v) BSA. Antibodies should be diluted in carbonate buffer (32 mM Na2CO3/16 mM NaHCO3) from 100 ng/µl stock solutions directly before use.