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% CO
2. 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% CO
2 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.
Ma R., Ma L., Weng W., Wang Y., Liu H., Guo R., Gao Y., Tu J., Xu T.L., Cheng J., Zhu M.X., Zhou A, & Li Y. (2020). DUSP6 SUMOylation protects cells from oxidative damage via direct regulation of Drp1 dephosphorylation. Science Advances, 6(13), eaaz0361.
