Angiogenic Matrigel assay in vitro

RH30 cells were cultured on six-well plates for 24 h in DMEM with 2% FBS. Subsequently, the conditioned media were collected and mixed with medium for endothelial cells supplemented with 2% FBS at a ratio of 1:1. For the Matrigel assay, 50 μL of growth factor-reduced Matrigel (BD Biosciences) was plated into a 96-well plate and incubated at 37°C for 30 min. HUVECs were detached and counted, and single-cell suspensions at a density of 10,000 cells per well in 200 μL of conditioned media and proper controls were plated on the Matrigel. Subsequently, endothelial tube formation was photographed 6 h after seeding. The formation of tubule-like structures was analyzed with Angiogenesis Analyzer for ImageJ (available online: https://imagej.nih.gov/ij/macros/toolsets/Angiogenesis%20Analyzer.txt). The numbers of junctions, master junctions, and nodes were calculated.