Conventional primary astrocyte culture

Primary astrocyte cultures were prepared from cerebral cortices of P2-P5 C57BL/6J mice. Cortices were dissected, stripped of meninges, and digested with 0.25% trypsin at 37 °C in Hank’s Balanced Salt Solution (HBSS) (Thermo Fisher) for 10 min. Trypsinization was stopped by the addition of astrocyte culture medium (DMEM/ F12 50/50 (Thermo Fisher) containing 25 mM glucose, 4 mM glutamine, 1 mM sodium pyruvate, and 10% FBS). Single-cell suspension of the digested tissue was obtained by repeated pipetting. Cells were seeded into a 75 ml flask at a density of ~4 × 10⁵ cells/cm² and cultured in astrocyte culture medium at 37 °C in a humidified 5% CO₂ incubator. Monolayers of glial cells were obtained 7–10 days after plating. To remove microglia, cultures were gently shaken, and the floating cells (microglia) were removed, resulting in more than 95% pure astrocytes. The remaining astrocytes were incubated for 72 h before being infected with lentivirus or other experiments. Before experiments, astrocytes were dissociated by trypsinization and then reseeded at 4 × 10⁵ cells cm² or 1.5 × 10⁵ cells per well in 24-well or 1.5 × 10⁶ cells per well in 6-well or 2 × 10⁵ cells cm² in Labtek imaging dish in DMEM F12 50/50 containing 10% FBS and 1% penicillin-streptomycin. At the start of each experiment, the medium was changed to regular DMEM without serum and antibiotics.