2.2. Primary rat astrocyte culture and identification

Postnatal 1–2-day Sprague-Dawley (SD) rats were provided by the Laboratory Animal Center of Jiangsu University. Protocols were approved by the animal ethics committee of Jiangsu University and carried out in accordance with established Guiding Principles for Animal Research.

Primary astrocyte cultures were prepared as described previously (Bai et al., 2015; Bai et al., 2016; Fang et al., 2016; Yang et al., 2018). Briefly, cerebral cortices were harvested and separated from the meninges under sterile conditions. The tissues were dissociated with trypsin for 15 min. Cells were suspended in fresh DMEM/F-12 supplemented with 10% FBS and 1% penicillin/streptomycin. After centrifugation at 1000 × g for 5 min, the cell pellets were resuspended and seeded on dishes. The cultures were maintained at 37°C in a humidified 5% CO₂–95% air atmosphere. Culture medium was changed after 24 h, and then replaced every 3 days. At confluence (10–14 days), cells that remained adhered to the flasks were astrocytes. The purity of the astrocyte cultures, ascertained by immunocytochemical staining with glial fibrillary acidic protein (GFAP), was > 95% (data not shown).