Protocol for isolating viable human central nervous system T cells

Brain-resident T cells act as sentinels, monitoring and supporting immune surveillance and homeostasis. Here, we present a rapid protocol for isolating viable T cells from post-mortem human brain tissue. We describe the process of extracting cells from multiple CNS compartments-including choroid plexus, leptomeninges, dura mater, cerebrospinal fluid, and parenchyma-as well as matched peripheral blood. We detail steps for achieving this through mechanical and enzymatic tissue dissociation, followed by density gradient centrifugation to isolate mononuclear cells for downstream applications. For complete details on the use and execution of this protocol, please refer to Hsiao et al.¹.