Ifnγ elispot kit plates

Mice bearing 10-day established B16^10%TRP1KO tumors were treated by Th1, Th9, or Th17 cell ACT as described above. Mice were sacrificed on day 20 after ACT and tumor tissues were minced and digested by tumor dissociation kit (Miltenyi Biotec). Each host immune cell subset in about 200 mg tumor tissues was isolated by a bead positive selection kit (CD8, NK1.1, or CD4). Isolated cells per 200 mg tumor tissues were cocultured with irradiated B16^TRP-1KO tumor cells on IFNγ ELISpot Kit plates (Mouse IFN-gamma ELISpot Kit, R&D Systems) for 48 hours following the manufacturer's instructions. The plates were imaged and evaluated by Cellular Technology Limited ELISPOT Analyzer.

B16^{20%TRP−1-KO} cells (1×10⁶) were s.c. injected into both flanks of B6 mice. TRP-1 T cells (5×10⁶) or TRP-1 T^10%MYXV cells (5×10⁶) were i.v. injected on day 7 when tumors reached ~7×6 mm. In some mice, free MYXV was i.t. injected only into the tumors on the left flanks. Mice were sacrificed on ~20 days after tumor inoculation and tumor tissues were minced and digested using a tumor dissociation kit (Miltenyi Biotec). Each host immune cell subset in about 200 mg tumor tissues was isolated by a bead positive selection kit (CD8⁺ or CD4⁺). Isolated cells per 200 mg of tumor tissues were cocultured with irradiated B16^TRP−1-KO tumor cells on IFNγ ELISpot Kit plates (Mouse IFN-gamma ELISpot Kit, R&D Systems) for 48 hrs following the manufacturer’s instructions. The plates were imaged and evaluated by a Cellular Technology Limited ELISPOT Analyzer.