Prolonged hypoxic conditions in vitro

Immortalized mouse brain microvascular endothelial cells from the cell line bEnd.3 derived from mice were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 100 U/ml penicillin and streptomycin (Thermo Fisher Scientific) and maintained in a humidified incubator (95% air/5% CO₂; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C. Cells were treated with Cobalt chloride (CoCl₂, 0,62.5,125,250, 375,500 µM, sigma, C8661) for 24 h when growing to 80% confluence in order to induce prolonged hypoxic conditions. ⁴⁵ In order to identify the optimal concentration of CoCl₂ solution, we measured HIF-1α expression and employed Cell Counting Kit 8 (CCK-8) reagent (1:10 dilution in medium; Dojindo, Kumamoto, Japan).