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  • Antigen-Specific T Cells
  • Antigen-Presenting Cells (APC)
  • Antigen
  • Culture Medium: We use X-VIVO™ 15, but other media may also support T cell activation
  • Mitomycin C


  1. 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.
  2. Add APC to 96 well U bottom plate at 20,000 cells per well (B-LCL) or 100,000 cells per well (PBMC).
  3. 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.
  4. Add T cells at 20,000 cells per well.
  5. 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.


  • Proliferation is a good measure of CD4+ cell proliferation, but CD8+ T cells do not seem to proliferate without addition of cytokines such as IL-2.
  • CD8+ T cell activation can be measured by cytokine production. Interferon gamma and TNF alpha are usually present after antigen stimulation.
  • If cytokine production is used as a read out, the antigen-presenting cells do not need to be inactivated.
  • Both cytokine and proliferation can be measured from the same assay, if desired.