Enzyme-linked ImmunoSpot (ELISpot) assay to quantify peptide-specific IFN-γ production by splenocytes in a mouse tumor model after radiation therapy.

To develop new effective therapeutic strategies for cancer patients, there is a need for extensive and precise insights into the mechanisms involved in the immune response to anti-cancer treatments. The enzyme-linked immunospot (ELISpot) assay is a rapid and reproducible technique that allows the detection of cytokine-producing antigen-specific T cells at the single cell level. This protocol describes an interferon gamma (IFN-γ) ELISpot method for measuring antigen-specific murine CD8⁺ T cells that produce IFN-γ, a marker of their activation and cytotoxicity. Splenocytes from tumor-bearing mice treated with radiation therapy were used as source of CD8⁺ T cells and were stimulated with a tumor-derived peptide. This method was facilitated by a ready-to-use assay kit and provides a tool to analyze the specificity, intensity, and kinetics of specific CD8⁺ T cells.