Isolation and culturing of primary mouse cortical astrocytes.

Cerebral cortices from postnatal mice 1–3 days old were aseptically dissected, and meninges were removed. The cortices were kept in HBSS and cut into small pieces with sharp blades. Tissue was then transferred to a 50-ml Falcon tube, trypsin was added to a final concentration of 0.25%, and tissue was incubated in a water bath at 37°C for 30 minutes. The sample was then centrifuged for 5 minutes at 300 × g, and the supernatant was decanted. Astrocyte plating medium (DMEM with high glucose) was added, and cells were dissociated by vigorous pipetting and again centrifuged at 400 × g for 5 minutes. The cells were resuspended in culture media (DMEM, high glucose + heat-inactivated 10% FBS, 1% Penicillin-Streptomycin) and seeded at a density of 3 × 10⁵ to 5 × 10⁵ cells per 75 cm² tissue culture flask precoated with poly-L-lysine and cultured at 37°C in a 95% air/5% CO₂ incubator. Culture medium was changed at 48 hours and then every 72 hours. Microglia were removed by rotation at 180rpm on an orbital shaker for 30 minutes when the astrocytes were confluent and overlaying microglia were detaching from the astrocyte layer. Media containing the microglia was aspirated, 20 ml of fresh astrocyte culture medium was added, and oligodendrocyte precursor cells were removed by shaking the flask at 240 rpm for 6 hours. The culture medium was again discarded, and the enriched astrocytes were reseeded into a new flask and further cultured at 37°C in 5% CO₂.