J. Clin. Invest. 118(1): 89-99 (2007)
Endothelial cell isolation and proliferation assay. Primary endothelial cells were isolated from adult mouse livers following mechanical mincing and collagenase (2 mg/ml) digestion for 30 min at 37°C. Cells were isolated by incubating for 30 min at 23°C with magnetic Dynabeads (Invitrogen) labeled with anti-CD31 antibodies and using a magnetic separator (53). Cells were then incubated in DMEM supplemented with 20% FBS, penicillin/streptomycin (100 U/ml), heparin (100 μg/ml), endothelial cell growth factor (100 μg/ml), 1× nonessential amino acids, 2 mM l-glutamine, 1× sodium pyruvate, gentamicin (58 μg/ml), and amphotericin B (25 ng/ml), termed “complete endothelium medium.” To assess the effects of VEGF-A on endothelial cell proliferation, cells from passage 1 or 2 were plated for 4 h in 96-well cell culture plates coated with fibronectin (5 μg/ml) at a density of 4,000 cells per well. The medium was then replaced with fresh complete endothelium medium containing 2% FBS and 0.5% BSA. Cells were incubated at 37°C for 48 h in the absence or presence of 50 ng/ml VEGF-A, prior to the addition of One Solution reagent (Promega) for 2 h. Optical density was then determined at 490 nm on a Molecular Devices microplate reader.
