2.6 Tube formation assays
The tube forming ability of HUVECs was determined by growth factor-depleted Corning® Matrigel®Basement Membrane Matrix(Corning, USA). Next, we added thawed matrix glue into the 96-well plate (70μl /well) and gelled matrix in the cell incubator for 30 min. Four groups of cells were cultured with different stimulating factors for 40 hours. Then, we collected cells from each group. Next, 100μl of cell suspension from each group (4 × 104 cell)was inoculated on the gelled matrix glue. For the normoxic+harmine group and the hyperoxia+harmine group, we added 100μl of medium containing harmine (6μmol/L) to pretreat the matrix glue for 30min before adding the cell suspension.The two groups stimulated by hyperoxia were given 95% O2 for 10 minutes. Finally, the four groups of cells were incubated in an incubator at 37℃ and 5% CO2 for 8h. Tube formation ability was then observed with an inverted optical microscope.