Primary microglia culture and adult brain microglia isolation

Mixed glia cells were prepared from the whole brain of neonatal mice at postnatal P1 and P2 and were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 20% fetal bovine serum (FBS; Gibco) at 37°C in a 95% O₂ and 5% CO₂ incubator for 8 to 10 days, and the medium was changed every 3 days. Microglia were isolated from the mixed glial cultures by mild agitation at 200 rpm for 6 hours in a rotary shaker at 37°C based on the distinct adhesive features of microglia and astrocytes. The obtained microglial cells were seeded into six-well plates in high-glucose DMEM supplemented with 20% FBS at a density of 1 × 10⁶ per well for 24 hours before further treatment. The purity of the adherent cells was verified by immunocytochemical staining, which indicated that more than 95% of the cells in the cultures were positive for the microglia-specific marker Iba-1 (1:500; Wako) (fig. S1H). A highly enriched population of microglia/macrophages was isolated from adult mice by Percoll density centrifugation using a protocol described previously (42). For the induction of a proinflammatory phenotype, LPS (100 ng/ml) and IFN-γ (20 ng/ml) were added to the microglial cultures for 24 hours. For the induction of an anti-inflammatory phenotype, IL-4 (20 ng/ml) was added to the culture for 24 hours (24).