Tube Formation Assay for Angiogenesis

A 24-well plate was coated with Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ, USA) mixed to EGM-2 1:1 on ice and incubated at 37 °C for 30 min to allow gelation to occur. As reported in [47 (link)], HUVEC cells were added to the top of the gel at a density of 2 × 10⁴ cells/well in the presence or not of the indicated treatments. Cells were incubated at 37 °C with 5% CO₂. After 12 hours, pictures were captured using EVOS^® light microscope (10×) (Life technologies Corporation, Carlsbad, CA, USA). The length of each tube was measured and the number of branches was calculated using ImageJ (NIH, Bethesda, MD, USA) (Angiogenesis Analyzer for ImageJ) software.