Protocol to isolate and enrich mouse splenic naive CD4(+) T cells for in vitro CD4(+)CD8αα(+) cell induction

Recent studies have shown that CD4(+)CD8αα(+) T cells are induced in the hypoxic environment of the small intestinal epithelium. Herein, we describe a protocol for CD4(+)CD8αα(+) T cell induction from freshly isolated naive CD4(+) T cells, including procedures for the isolation and enrichment of mouse splenic T cells. In addition, we present an approach that can induce more CD4(+)CD8αα(+) T cells by artificially creating a hypoxic environment in vitro. For complete details on the use and execution of this protocol, please refer to Harada et al. (2022).