2.10. Matrigel assay (tube formation)

EC were cultured in M200 medium containing 5% FBS and 5 mg/ml of EC growth factor (ECGF, Millipore). The effects of leptin and inhibitors of VEGFRs and Notch on EC ability to form tube-like structures in vitro were determined by Matrigel Matrix assays (BD Sciences). VEGF was used as positive a control. Growth factor-reduced Matrigel was plated onto 96-well plates (200 µl/well) and incubated at 37°C for 30 min. HUVEC were seeded in the Matrigel-coated plates at 10,000 cells/well, and treated with leptin (1.2 nM), SU5416 (5 µmol/l), and DAPT (5 µmol/l) in 1% reduced FBS medium. After 8 h, the cells were washed twice in PBS and fixed in 4% paraformaldehyde. Images of tube formation were captured at 10× using an inverted microscope. Quantitative determination of HUVEC tube formation was carried out using Image Pro Plus software. The visible tubes were counted using the software, and the analyses of vessel average length and area (mm²) were performed.