Matrigel-Based Endothelial Tube Formation Assay

Pipette tips, pre-chilled, were used to draw 50 μL of Matrigel assay (BD Biosciences, Bedford, MA, USA) uniformly across the base of a 96-well plate. Following distribution, 96-well plates were situated in a 37 °C cell culture incubator for 30 ~ 60 min for solidification. Test cells were digested, and cell suspensions were prepared. The cells were then counted and adjusted to 25,000 cells and 200 μL of cell suspension per well was injected into a 96-well plate containing three matrix wells. After 6 h, cells were observed using an inverted light microscope and imaging system. The total branch length of endothelial cells in each group was quantified using vascular analysis software (Angiogenesis analyze). The tube formation assay was repeated thrice. The assay was conducted as previously described^{24 (link)}.