Matrigel-based Angiogenesis Assay with KH-204 and Li-ESWT

Two hundred microliters of BD growth factor reduced (GFR) Matrigel™ Basement Membrane Matrix (BD Biosciences, Franklin Lakes, NJ, US) was added to a 24-well plate and incubated at 37 °C for 30 minutes, then HUVECs (1×10⁵ cells/well) were incubated in one plate coated with Matrigel in endothelial growth media (EGM) and on a plate coated with endothelial basal media (EBM) (18 (link)). KH-204 (50 µg/mL) was added to the plates for two hours in the KH-204 group and KH-204/Li-ESWT groups (10 (link)). The other group received an equal amount of medium as a control. After cell adherence, Li-ESWT was performed on the Li-ESWT group and KH-204/Li-ESWT group cells for 20 min. After 12 hours of treatment, the cells were stained using calcein AM dye (2 µg/mL). The formation of cell tubules was observed with an Axio200 fluorescence microscope (Zeiss, Oberkochen, Germany). Images were traced and skeletonized using Image and the angiogenesis tool. The total number of meshes, nodes, and junctions were quantified for each skeleton.