Cortical Neuron Culture and OGD/Reox Assay

Cortical cultures were prepared from embryonic day 13.5 (E13.5) C57BL/6 mouse embryos by dissection, trypsin treatment, and mechanical dissociation. Cells were plated on poly-l-lysine–coated dishes at a density of 3 million per 6-cm dish and 0.15 million per well for 24-well plates in Neurobasal medium (Gibco) containing 2% B27 (Gibco) and 1% GlutaMAX (Gibco) and incubated at 37°C in humidified air supplemented with 5% CO₂. Cortical neurons were then fed every 4 days with this serum-free medium until experimental usage at 14 days in vitro (DIV). The OGD/reox was performed as described previously (59 (link)) with minor modifications. Cortical neurons grown in 6-cm dishes or grown on glass coverslips in 24-well plates were transfected with or without Flag-DUSP6 plasmid using NeuroPORTER transfection kit (NPT01, Sigma-Aldrich), and cells were incubated at 37°C in 5% CO₂ for 36 hours following transfection. Then, neurons were subjected to 2-hour OGD and 24-hour reoxygenation. Neuronal cell lysates were used for Western blotting, and neurons grown in 24-well plates were used for TUNEL staining.