Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system

Microglia are the resident macrophages of the central nervous system and are highly sensitive to environmental cues, making primary culture challenging. Here, we present a protocol for isolating microglia from adult mouse brains using magnetic-activated cell sorting (MACS). The protocol includes brain sample collection, enzymatic tissue dissociation, step-by-step MACS-based microglia isolation, and the establishment of primary microglial cultures. We validate isolated cells using flow cytometry and quantitative real-time PCR (real-time qPCR) to assess microglial purity. • Protocol for isolating and culturing primary microglia from adult mouse brain • Steps to maximize microglial yield using magnetic-activated cell sorting • Procedures for establishing viable primary microglial cultures from adult brain tissue • Guidance on validating microglial purity and culture suitability Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Microglia are the resident macrophages of the central nervous system and are highly sensitive to environmental cues, making primary culture challenging. Here, we present a protocol for isolating microglia from adult mouse brains using magnetic-activated cell sorting (MACS). The protocol includes brain sample collection, enzymatic tissue dissociation, step-by-step MACS-based microglia isolation, and the establishment of primary microglial cultures. We validate isolated cells using flow cytometry and quantitative real-time PCR (real-time qPCR) to assess microglial purity.