Lipid supplement

Cultures of hCMEC/D3 cells (≤P35) were grown in MCDB 131 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 5% FBS, GlutaMAX (100×, Life Technologies, Carlsbad, CA, USA), lipid supplement (100×, Life Technologies, Carlsbad, CA, USA), 10 µg/mL ascorbic acid, 550 nM hydrocortisone, 100 µg/mL heparin, 1 ng/mL basic fibroblast growth factor (bFGF, Roche, San Francisco, CA, USA), 2.5 µg/mL insulin, 2.5 µg/mL transferrin, 2.5 ng/mL sodium selenite (ITS), and 50 µg/mL gentamicin [84 (link)]. All of the plastic surfaces were coated with 0.05% collagen in sterile distilled water before cell seeding and the medium was changed every 2 days. Before each experiment, the medium of hCMEC/D3 cells was supplemented with 10 mM LiCl for 24 h to improve the barrier properties [85 (link)].

The hCMEC/D3 human brain microvascular endothelial cell line was obtained from Merck Millipore (Germany). To maintain the cells’ brain endothelial-like features, we used cells under passage number 35 [16 (link)]. Cells were grown in dishes coated with rat tail collagen and maintained in an incubator at 37°C with 5% CO₂. The basal medium used was MCDB 131 (Pan Biotech, Germany) supplemented with 5% fetal bovine serum, GlutaMAX (100 ×, Life Technologies, USA), lipid supplement (100 ×, Life Technologies, USA), 10 μg/ml ascorbic acid, 550 nM hydrocortisone, 37.5 μg/ml heparin, 1 ng/ml basic fibroblast growth factor (Roche, USA), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium supplement (100x, PanBiotech), 10 mM HEPES, and gentamycin (50 μg/ml). We changed the medium every two or three days. When the cultures reached almost 90% confluence, we passaged them to rat tail collagen-coated 96-well plates (E-plate, Agilent, USA) for impedance measurement assays, cell culture inserts (0.4 μm pore size, cellQUART, Sabeu, Germany) for barrier integrity assays and 96-well black-wall plates (Corning, USA) for ROS measurement. Before each experiment, the medium was supplemented with 10 mM LiCl for 24 hours to improve BBB properties [17 (link)]. For further characterization and gene expression studies please see [15 (link)–17 (link), 24 (link)].