Hypoxia-Induced Injury in bEnd.3 Cells

bEnd.3 cells were rinsed twice with Earle’s Balanced Salt Solution (EBSS; 116 mM NaCl, 5.4 mM KCL, 26.2 mM NaHCO₃, 1.8 mM CaCl₂, 1 mM NaH₂PO₄H₂O, 0.8 mM MgSO₄, and 0.01 mM glycine), and the media were replaced with glucose-free EBSS or EBSS supplemented with 5.5 mM glucose for OGD stimulation or control treatment, respectively. Cell plates were placed in a hypoxia chamber (Billups-Rothenberg Inc., San Diego, CA, USA), and the air was replaced with OGD gas (95% N₂ and 5% CO₂) by flushing. Cells were exposed to the OGD condition for 6, 12 or 18 h at 37 °C to measure the cell viability. After selecting 18 h OGD as the ischemic insult, cells were exposed to 18 h OGD for the other experiments. Oxygen depletion in the chamber was monitored using BD GasPak™ Dry Anaerobic Indicator Strips (BD, Franklin Lakes, NJ, USA). For autophagic inhibition experiments, bEnd.3 cells were treated with 3-methyladenine (3-MA; Sigma-Aldrich) for 1 h prior to OGD stimulation. The concentration of 3-MA was selected according to the previous reports [16 (link), 20 (link)]. Treatment with 3-MA was continued during OGD exposure.