PMC:3317373 / 2939-4307 JSONTXT

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After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). 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