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).
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