Poly d lysine pre coated 96 well plates

Brains were removed from embryonic day 15 C57BL/6 mice [36 (link)] and maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) with 25μM of 2-mercaptoethanol at 4°C during dissection. Cortices were dissected and dissociated in trypsin (0.05% with EDTA) and DNAse (10 μg/ml) and then resuspended in neurobasal (NB) medium with 2% B27 and 1mM glutamine supplement (Gibco, Rockville, MD). The cells were plated onto poly-D-lysine precoated 96-well plates or 35-mm dishes (Corning Life Sciences, Tewksbury, MA) at a density of 1 × 10⁵ cells/cm². The cultures were kept at 37°C in a humidified incubator with a 5% CO₂ atmosphere. Three days after plating, half of the medium was replaced with the NB medium with 5-Fluoro-2’- deoxyuridine (4 μg/ml) and Uridine (20 μg/ml) to inhibit growth of non-neuronal cells. The neurons were used on 7–8 day in vitro (DIV) for oxygen-glucose deprivation (OGD) experiments.