Transfection of Effector Jurkat E6.1 Cells

Jurkat E6.1 T cells were cultured and passaged at least once at 0.3×10⁶cells/mL before transfection in RPMI supplemented with 10% FBS, 1% L-glutamine and 1% penicillin streptomycin. Cells were pelleted and washed twice in Opti-MEM prior to resuspension at 5×10⁷cells/mL. Jurkat cells were electroporated using Gene Pulser Xcell Electroporation System (Bio-Rad Laboratories, Inc.) using the protocol SQR wave, 230V, 25ms, 1 pulse with CE module (V, % droop) in 200µL of Opti-MEM with four plasmids: CD8alpha (5mg); TCR alpha (3mg); TCR beta (3mg); and NFAT luciferase (10mg). Electroporated cells were rested for 10 minutes prior to a 24-hour incubation in 5.8mL RPMI (no phenol red) supplemented with 10% FBS and 1% L-glutamine at 37°C and 5% CO₂.