Ifn γ elispot

To measure the splenocytes responding to tumor antigens, IFN-γ ELISPOT (551083, BD Bioscience) was performed following the manufacturer’s instruction. Briefly, when average tumor volume reaches about 100 mm³, CT26.WT tumor-bearing mice were intratumorally injected with PBS or KLS-3020 at a dose of 8 × 10⁷ TCID₅₀ three times every other day. Twenty-eight days after the last virus injection, the spleen was harvested and homogenized using a cell scrapper on a 70-μm cell strainer. The homogenates were washed with complete medium (DMEM with 10% FBS, 1% AA, and 2 mM L-glu) and collected in a single conical tube. After centrifuging at 400 × g for 5 min, the pellets were suspended with RBC lysis buffer and incubation for 1 min at RT. Then the cells were centrifuged at 400 × g for 5 min, suspended with complete medium, and passed through a 70-μm cell strainer to remove the cell debris. Then the 1 × 10⁵ splenocytes were co-incubated with 3 × 10⁴ CT26.WT cells to activate the splenocytes with CT26.WT-specific immune response. Eighteen hours after the incubation, the cells were colorized following the application of biotinylated anti-IFN-γ antibody, streptavidin-HRP, and AEC substrates. The numbers of the spot were counted with an ELISPOT reader (Eli.Scan+, AELVIS).