Mouse primary microglia isolation and culture

Mouse primary microglia were purified according to the protocol previously described with minor modifications ^{8, 61, 62}. In brief, mice brain was dissected from neonatal mice (1-3 days) and washed three times with 5 mL ice-cold wash media (low-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin-streptomycin) to remove blood. Next, both the olfactory bulb and cerebellum were removed. The meningeal layer was carefully stripped to avoid contamination of the monocytes in the blood vessels and damage to the cortices. The cortices were digested with 3 mL 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Life Technologies, #25200072) for 20 min and dispersed to a single-cell level by passing through a cell strainer (70 µm) following the stop digestion by DMEM containing 10% FBS. The cell suspension was centrifuged at 500 × g for 5 min at 4 °C and resuspended with growth medium then cultured at 37 °C in humidified 5% CO₂ and 95% air on poly-D-lysine (10 µg/mL) (Beyotime, #C0312)-precoated 75 cm² cell culture flasks. The medium was half-replaced every 4-5 days. After the cells reached confluence (8-10 days), the astrocytes and microglia were isolated by mild trypsinization with 0.05% Trypsin-EDTA (Life Technologies, #25300054) for 5 min ^{8, 61}. In detail, treatment of the confluent mixed glial cultures with 0.05% Trypsin-EDTA resulted in the detachment of an intact layer of cells containing almost all of the microglia and leaving behind highly enriched astrocytes. Over 95% of the microglia cultures obtained by the digestion of trypsinization were positive for IBA-1. The cellular yield is 4.0×10⁵ microglia/neonatal mouse. More than 85% of the remaining cells were astrocytes, as determined by staining with GFAP (not shown). After the isolation procedure, the attached microglia were allowed to recover for 24 h, and the cells were further plated as required for the specific experiments. To obtain sufficient primary cells, we collected the primary cells from five mice as a mixture to seed plate.