HUVEC network formation with VEGF-A

Network formation in the ETFA was carried out by seeding HUVEC (10⁵ cells/well) on Matrigel (250 µl/well) into a 24-well plate for 24 h at 37 °C with 5% CO₂. Cells were suspended in EGM2-MV medium without VEGF-A (control condition), or complemented with 5, 10, 25 or 50 ng/ml VEGF-A. Sunitinib, a multitargeted tyrosine kinase inhibitor was added (5 nM or 25 nM) to culture medium as an angiogenesis inhibitor. Five pictures per well (center of the well and four cardinal points) were taken at time 24 h using a camera Nikon D5300 associated to an inverted microscope Nikon Eclipse TS100 using a 4× objective (NA 0.13) in phase contrast mode without fixation. For statistical analyses, three wells were seeded per conditions.