Protocol for generating highly enriched sex-specific primary cortical astrocytes from individual mouse pups

Here, we present a protocol for generating highly enriched primary cortical astrocytes from individual neonatal mouse pups. We describe steps for mechanically dissociating cortical tissue, culturing in coated flasks, and removing oligodendrocyte progenitor cells through mechanical shaking and selective pharmacological depletion of microglia. We then detail procedures for PCR-based analysis of tail-biopsy DNA to determine sex and genotype to genetically define astrocyte cultures. This protocol enables the investigation of sex- and genotype-specific mechanisms while avoiding the confounding effects of pooled tissue.