A roadmap for enriching rare cell populations from human post-mortem brain, demonstrated by 100% microglial purity.

Understanding brain disease requires studying the specific cell types that drive each condition: microglia in Alzheimers, dopaminergic neurons in Parkinsons, motor neurons in ALS, and many others. For most of these rare populations, protocols to isolate them from human post-mortem brain at the required purity have never been developed. The shared hurdles are reliable nuclear markers, compatible fluorescent dyes, antibody host-species cross-reactivity, and achieving pure rather than merely enriched populations. Here we present a generalisable roadmap for developing fluorescence-activated nuclear sorting (FANS) protocols for rare cell populations in human brain. We demonstrate it on microglia, reaching 100% purity in cortex and 98% in cerebellum, with the cortex sort simultaneously yielding astrocytes at 93% purity. The roadmap tackles each hurdle including an on-bench antibody labelling step that expands fluorescence channels without cross-reactivity problem and an affordable single-nucleus RNA-seq validation workflow, giving labs a pathway for enriching any rare cell type reliably.