T-cell activation assay protocol

Cells were harvested from culture washed with R0 (as for R10 but with no FBS) and rested overnight in R5 media (as for R10 but with 5% FBS). Resting ensured minimal background activation of the T-cells. On the day of activation assay, cells were harvested, and 2–5 × 10⁴ cells were incubated with 30 µM TAPI-0 (Sigma-Aldrich) (54 (link)) anti-TNF-PE-Vio770™ (clone cA2, Miltenyi Biotech) and anti-CD107a-PE (clone H4A3, BD Biosciences) Abs in well(s) of a 96U-well plate. For peptide presentation, autologous LCLs were used for clonal T-cells, and T-cell to T-cell presentation used for T-cell lines. The cells were incubated for 4–5 h at 37°C then stained with Vivid and Abs for CD3 and/or CD8, as above.