Brain Microvascular Endothelial Cell Culture

Human brain microvascular endothelial cells (HBMECs), cell culture media, and growth supplements were obtained from Sciencell (Carlsbad, CA, USA). Primary antibodies against phospho-AMPKα (Thr-172), AMPKα, GAPDH, and all secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against occludin and anti-claudin-5 were from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies against the NAD(P)H oxidase subunits (p47^phox, p67^phox) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC-dextran was from Sigma–Aldrich (St. Louis, MO, USA). A stock solution of LPS (Escherichia coli O111:B4; Calbiochem, Germany) was prepared in PBS. The concentration of LPS was selected to mimic sepsis [17 (link)].