Protocol for the isolation of mouse senescence-associated CD4⁺ T cells using flow cytometry and functional assays

Senescence-associated (SA) CD4⁺ T cells, which increase with age, may underlie the development of autoimmunity and chronic inflammation, but their pathological function remains understudied. Here, we present a protocol to isolate CD153⁺ SA-T cells and evaluate their characteristic responses upon T cell receptor stimulation. We describe steps for the isolation of CD153⁺ SA-T cells using flow cytometry and in vitro culture with stimulatory antibodies against CD3, CD28, and CD153. We then detail the assessment of the proliferation capacity and cytokine production. For complete details on the use and execution of this protocol, please refer to Fukushima et al. (2022).¹.