Hypoxia-reoxygenation in brain endothelial cells

Human brain microvascular endothelial cells were purchased from Applied Cell Biology Research Institute (Kirkland, WA, USA). OGD/R was performed as previously described [14 (link)]. Confluent cells were washed and replaced with glucose-free Dulbecco’s Modified Eagle’s Medium (DMEM). Cells were then incubated with AnaeroPack (AN0025A; Thermo Fisher Scientific) for 6 h. After OGD, cells were reperfused by placing them in endothelial basal medium 2 (CC-3156; Lonza, Walkersville, MD, USA) containing vehicle or CU06-1004 (10 μg/mL) and were incubated in a 95% air/5% CO₂ for 16 h. After 16 h, immunofluorescence staining was performed.