Primary Neuron Isolation from Mouse Cortex

Primary neuron cells were isolated from the cerebral cortex of 16-day C57BL/6 mouse embryos as described previously [70 (link)]. Briefly, the cerebral cortex was separated from the brains and then dissociated with 0.25% trypsin (wt/vol) at 37°C for 30min. After centrifugation, the cells were resuspended in DMEM containing 5% FBS (Gibco) and subsequently passed through a 70 μm filter. After cell counting, the cells were seeded into 12-well plates (SORFA Life Science Co., Ltd, Beijing, China) that had been pretreated with poly-D-lysine and laminin (10 μg/ml) (Sigma, P7280). The cell supernatants were discarded at 5 h after seeding and replaced with Neurobasal plus Medium (Gibco, A3582901) containing 2% B-27 Plus (Gibco, A3582801). Then replace the medium every two days until 5–6 days post seeding for experiments.