Quantifying Tumor-Specific T-Cell Response

Spleens from DC–tumor cell fusion vaccine‐treated mice were isolated 10 days after the last DC–tumor cell fusion vaccine injection. Splenocytes were isolated from the spleens as described previously. To stimulate splenocytes, B16‐F10 melanoma cells were treated with 15 μg·mL⁻¹ mitomycin C (Nacalai Tesque Inc.) for 45 min. Splenocytes harvested from vaccine‐treated mice and mitomycin C‐treated B16‐F10 melanoma cells were mixed at a ratio of 10 : 1 and co‐cultured for 48 h. Nonadherent splenocytes were collected, and ELISpot assay was performed using the Mouse IFN‐γ Development Module (R&D Systems, Minneapolis, MN, USA) and the ELISpot Blue Color Module (R&D Systems). The numbers of IFN‐γ‐secreting cells were subsequently counted.