Human anti cd3 antibody clone sk7

NY-ESO-1 and MART-1 T cells were used for co-culture assays. Two days prior to co-culture, cells were thawed in T cell media containing 3 U/mL DNAse (Genentech Inc., South San Francisco, CA) overnight. Tumour cells were seeded at specific density on this day in the same media as the T cells. T cells were then cultured in T cell media containing 300 IU/mL interleukin-2 (IL2) for 24 hours. T cells were co-cultured with tumour cells at various Effector: Target (E:T ratios) for varying time periods. To reduce T cell killing activity and enrich for resistant tumour cells during the recovery phase, T cells were removed by careful 2x phosphate buffered saline (PBS) washes following the addition of D10 media without IL2. At the end of recovery phase of co-culture, tumour cells were detached using trypsin (Invitrogen) and washed twice with PBS. Tumour cells were then stained with fixable Live/Dead dye (Invitrogen) followed by human anti-CD3 antibody (clone SK7, BD) in FACS staining buffer (PBS + 0.2% BSA). Cell counts were measured using CountBright Absolute Counting Beads (Invitrogen) by FACS.

Two days prior to co-culture, T cells were thawed in T cell media containing 3 U/mL DNase (Genentech Inc.) overnight. Tumor cells were seeded at desired density on this day in T cell media. After 24 h, T cells were cultured in T cell media supplemented with 300 IU/mL IL-2 for 24 h. T cells were co-cultured with tumor cells at various effector: target (E:T) ratios for specified durations. After co-culture, T cells were removed by washing tumor cells with PBS and tumor cells were detached using trypsin. Cells were stained with fixable Live/Dead dye (Invitrogen) followed by human anti-CD3 antibody (clone SK7, BD) in FACS staining buffer (PBS + 0.2% BSA). Cell counts were normalized with CountBright Absolute Counting Beads (Invitrogen) by FACS.