Culturing Primary Human Brain Microvascular Endothelial Cells

hBMVECs were grown in DMEM/F12 media (Invitrogen) containing 10 mm HEPES, 13 mm sodium bicarbonate (pH 7), 10 mmol/l L-glutamine and supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin and streptomycin (100 μg/ml each, Life Technologies), 1% amphotericin (Caisson Labs), endothelial cell growth supplement (ECGS; 50 mg/ml; BD Bioscience), heparin (100 mg/ml; Sigma-Aldrich Co, Ltd; Abdul Muneer et al., 2012 (link), 2018 (link)). hBMVECs were plated on rat tail collagen Type I (0.1 mg/ml) coated six-well Bioflex culture plates (Flexcell International Corp) at a density of 250,000 cells/well (Patel et al., 2017 (link); Abdul-Muneer et al., 2018 (link)). The cells were fed by changing the medium every 2 d and grown until tight monolayers were formed in ∼6–8 d.