Falcon tissue culture dish

Primary astrocyte cultures were prepared from 1-day-old Sprague-Dawley rat brains (Animal Experiment Center, Southern Medical University, Guangzhou, China), as previously described (11 (link),12 (link)). Ethics approval was granted by the Medical Ethics Committee of Southern Medical University, Guangzhou, China. Briefly, a total of 12 rats (n=3/group) were deeply anesthetized with a mixture of ketamine and xylazine (70:6 mg/kg, intramuscular injection) and then decapitated, the striatums were dissected, and the meninges were removed. The cerebral hemisphere was freed of the meninges and cut into 1 mm³ cubes in Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Fresno, CA, USA). The tissue was dissociated by vortex mixing for 60 sec, and the cell suspension was passed through 70 and 20 µm sterile mesh nylon filters. A volume of cell suspension containing approximately 4.5×10⁵ cells was seeded in a 35 mm² Falcon tissue culture dish (Corning, Albany, NY, USA). Primary astrocytes were cultured in DMEM containing 10% fetal bovine serum (FBS) and 0.15% penicillin-streptomycin reagent (both from Gibco Life Technologies) at 37°C with 5% CO₂. The medium was changed twice a week. Cultures of at least 4 weeks were used in the experiments.