Culturing Primary Cortical Neurons from Mice

Primary cortical neurons were prepared from C57BL/6J mouse embryos (Charles River) of either sex on embryonic day 17. Cerebral cortices were dissected and enzymatically dissociated using trypsin with EDTA (Thermo Fisher Scientific; 10 min), mechanically dissociated in Minimum Essential Media (MEM; Fisher) supplemented with 0.6% glucose (Sigma) and 10% FBS (Hyclone) and stained to assess viability using Trypan Blue (Sigma). Neurons were plated on coverslips (Matsunami Inc, 22 mm). A total of 50,000 neurons were plated as a ‘spot’ on the center of the coverslip to create a small, high-density network. Neurons were cultured in standard growth medium (glial conditioned neurobasal plus medium [Fisher] supplemented with GlutaMAX [Gibco], and B27 plus [Invitrogen]), and half of the media was exchanged 2–3 times a week until the experiment endpoints. No antibiotics or antimycotics were used. Cultures were maintained in an incubator regulated at 37^οC, 5% CO₂, and 95% relative humidity as described (Valdez-Sinon et al., 2020 (link)). Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.