Isolation and Culture of Primary Cortical Neurons

Primary cortical neurons were isolated from cerebral cortices of embryonic day 13–14 mouse embryos (Charles River Laboratories) as previously described [93 (link)] with modifications. Briefly, pregnant female mice were euthanized between embryonic days 13 and 14, and embryonic cortices were dissected and enzymatically dissociated using a papain dissociation kit (Worthington Biochemical, LK003178). Neurons were plated on poly-D-lysine-coated 384- (Corning Life Sciences, 354,663) and 24-well plates (Falcon, 08-772-1) at densities of 1 × 10⁴ and 2.5 × 10⁵ viable neurons, respectively. The neurons were maintained at 37 °C with 5% CO₂ in Neurobasal medium (Life Technologies, 21,103–049) supplemented with 1% GlutaMAX (Thermo Fisher Scientific, 35,050–061), 2% B-27 supplement (Life Technologies, 17,504–044), and 1% penicillin/streptomycin (Life Technologies, 15,070–063). Primary cortical neurons were grown for 5 days before receiving described drug treatments and analysis.