Primary Rat Cortical Cultures: Preparation and Maintenance

Primary rat cortical cultures were prepared from embryonic day 18 Sprague-Dawley rat embryos (Charles River Laboratories, Seattle, WA). Brains were isolated, and dissected cortices were incubated for 40 min in DMEM +0.027% trypsin as described previously (Wilcox et al. 1994 (link)). Cells were then washed in saline, triturated, resuspended in neurobasal media supplemented with B27, and plated on poly-L-lysine-coated 6-well (9.4-cm² growth area) or 24-well (1.9-cm² growth area) plates (USA Scientific, Ocala, FL) at a concentration of 500,000 cells/ml. Cultures contained approximately 90% neurons and 10% astrocytes/glia and were maintained in neurobasal media supplemented with B27 at 37 °C with 5% CO₂ as described previously (Gannon et al. 2017 (link); Akay et al. 2011 (link)). On 10 days in vitro (DIV), 20% fresh media was added. Cells were treated on DIV 14–16.