In Vivo IFN-γ Elispot Assay

IFN-γ Elispot assays were conducted using the Mouse IFN-gamma ELISpot Kit (XEL485, R&D Systems) as according to the manufacturer’s instructions.49 (link) Spleens were collected on day 8 post-treatment. Spleen cells suspended in Roswell Park Memorial Institute (RPMI)-1640 Medium (supplemented with 10% FBS, 10 ng/mL mIL2, and 1 µM 2-mercaptoethanol) were loaded to the 96-well plates (1×10⁵ spleen cells per well). Antitumor cell immunity was analyzed by loading live tumor cells (1×10⁴ tumor cells per well) into the spleen cell preseeded wells. Antitumor antigen immunity was analyzed by loading tumor antigen peptides (Trp2 _180–188 or gp100 _25–33, a final concentration of 5 µM for each peptide) to the spleen cell preseeded wells. After tumor cells, peptides, or IL12s were loaded, the plates were cultured at 5% CO₂ and 37°C for 3 days before spots were developed and counted. Phytohemagglutinin P (inh-phap, Invivogen) (100 µg/mL) was added to the positive control wells.