Culturing Human Endothelial Cells

Immortalised human bone marrow ECs (HBMECs), and human cerebral microvascular ECs (HCMEC/D3s) were both donated by Prof B. B Weksler, Cornell University, New York and support leukocyte transendothelial migration studies (37 –39 (link)). HBMECs were maintained in DMEM-F12 (Lonza, Wokingham, UK) supplemented with 10% FBS (Invitrogen, Paisley, UK) and HCMEC/D3s in rat tail collagen type 1 (R&D Systems, Abingdon, UK) coated flasks (150 mg/ml in dH₂O) using EBM-2 (Lonza) containing 1% penicillin-streptomycin (Invitrogen), 1% chemically defined lipid concentrate (Invitrogen), 1 ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich), 1.4 μM hydrocortisone (Sigma-Aldrich), 5 μg/ml ascorbic acid (Sigma-Aldrich), 10 mM HEPES (PAA Laboratories, Yeovil, UK), 5% FBS and 10 mM lithium chloride (Merck, Feltham, UK). All ECs were incubated at 37°C in a humidified incubator containing 5% CO_2. Cells were grown to 70% confluence before being used in the following experiments.