Isolation and Ex Vivo Testing of CD8<sup>+</sup> T-Cell Division and Activation Using Mouse Splenocytes

This protocol describes an ex vivo co-culture method to assess CD8⁺ T-cell activation, proliferation, and cytotoxic potential using bulk splenocytes isolated from immunocompetent mice. Mouse splenocytes are stimulated with anti-CD3 and anti-CD28 antibodies to activate CD8⁺ T cells, which are then co-incubated with either cancer cells or cancer cell-derived conditioned media (CM) to evaluate tumor-driven modulation of immune cell functions. The use of unfractionated splenocytes preserves physiological cell-cell interactions, eliminating the need for exogenous interleukin (IL-2) and bypassing flow sorting, which simplifies the workflow and reduces experimental variability. CD8⁺ T-cell responses are measured via flow cytometry, using markers of proliferation (CFSE dilution), activation (CD69), and effector function (Granzyme B and IFNγ). Additionally, immune-mediated tumor cell death is evaluated by Annexin-V/7-AAD staining. Together, this experimental platform supports the investigation of both cell contact-dependent and contact-independent mechanisms of immune cell modulation in a cost-effective and reproducible setting. Key features • Enables isolation and stimulation of splenocytes to assess CD8⁺ T-cell responses to cancer cells or their secreted factors. • Supports evaluation of CD8⁺ T-cell activation, proliferation, and effector function by flow cytometry. • Allows functional assessment of tumor-driven suppression of T-cell activation in co-culture or conditioned media. • Measures cancer cell death resulting from interactions with activated CD8⁺ T cells in splenocyte co-cultures.