Cortical Neuron Culture and Oxygen-Glucose Deprivation

Cortical cells were harvested from embryonic day 17 Wistar rats (Charles River) according to a published protocol with minor modifications [18 (link)]. In brief, dissociated cortical cells at a density of 3 × 10⁷ cells/mL were cultured in microfluidic chambers (AXIS™ Axon Isolation Device, Millipore, Billerica, MA) in neurobasal medium with 2% B27, which included serum albumin, corticosterone, insulin, and progesterone (GIBCO, Grand Island, NY). Uridine and 5-fluorodeoxyuridine were added for 6 days to kill astrocytes. After 7 days in vitro, the medium was changed to Ca2⁺- and Mg2⁺-free Hanks’ balanced salt solution, and cultured neurons were challenged with OGD for 3 h. After OGD, cultured neurons were incubated in neurobasal medium containing 2% B27 for 96 h. Ten to 12 embryos were gathered and prepared for primary neuronal culture, and each experiment was counted as the number in vitro.