Astrocyte Oxygen-Glucose Deprivation and Reoxygenation

Rat brain cortex astrocytes were subjected to OGD/R as described previously [28 (link),54 (link)]. Briefly, cells were seeded at a density of either 5 × 10³ cells cm⁻² (low cell density) or 1 × 10⁴ cells cm⁻² (high cell density) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) with no glucose supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (P/S; Gibco, Waltham, MA, USA) and 1% or 10% (v/v) fetal bovine serum (FBS; Gibco) in an incubator filled with 1% O₂, 5% CO₂, and 94% N₂ at 37 °C. After 4 h, oxygen-glucose deprivation was terminated by changing the medium to DMEM with high glucose (Gibco) supplemented with 10% (v/v) FBS and P/S under normal conditions (37 °C with 95% air and 5% CO₂) for 20 h. Cells in the normal group were cultured in DMEM with high glucose supplemented with 10% (v/v) FBS and P/S under normal conditions.