In Vitro Cytotoxicity Assay Protocol

IFN-γ ELISpot assays were performed using the ELISpot kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the instructions provided by the manufacturer, and in vitro cytotoxicity assays were performed based on previous studies [33 (link)]. Simply put, Lewis cells were used as target cells and labeled with antigen peptides (final concentration: 5 μg/mL) or without antigen peptides and incubated for 2 h. Target cells incubated with peptides were stained with high concentrations (5 μM) of CFSE, while non-peptide-labeled target cells were stained with low concentrations (0.5 μM) of CFSE. Target cells stained with high and low concentrations of CFSE were evenly mixed 1:1 (a total of 1 × 10⁵ target cells), and then different numbers of spleen cells (effector cells) from vaccinated mice were mixed with target cells in different ratios. Effector cells and target cells were incubated at 37 °C for 8 h, and cytotoxicity was analyzed by flow cytometry, which detects a decrease in the percentage of specific target cells.