- Inactivate antigen-presenting cells with mitomycin C.
- If B-LCL are used as APC, adjust the cell concentration to 2 x 106 per mL in PBS. If PBMC or other non-transformed cell type is used, the cell concentration can be increased to 5 x 106 per mL in PBS.
- Add mitomycin C to a final concentration of 50 ug/mL for transformed cells, such as B-LCL, or 25 ug/mL for PBMC and other non-transformed cells.
- Incubate at 37°C for 45 minutes.
- Wash the cell three times with PBS using at least 10 mL per wash.
- Add APC to 96 well U bottom plate at 20,000 cells per well (B-LCL) or 100,000 cells per well (PBMC).
- Add antigen at a range of concentrations. Each cell line has an optimum antigen concentration. Data given in the Certificate of Analysis will provide an appropriate range.
- Add T cells at 20,000 cells per well.
- Incubate at 37°C, 5% CO2 for 1–4 days. If cytokine production is being measured, collect medium for testing after 24–48 hours. If proliferation is being measured, we have found day 4 to be the best.