Endothelial Cell Tube Formation Assay: An In Vitro Model for Angiogenesis

Endothelial cell tube formation assay is one of the most widely used and reliable methods for studying in vitro angiogenesis. Endothelial cells plated over a basement membrane extract and subjected to angiogenic factors in conditioned medium, form a rapid and quantifiable tube network within hours. Tube formation is sustained for 18–24 h, after which time apoptosis occurs and tube networks disintegrate. The tube network can be imaged using a phase contrast microscope, or upon Calcein-AM treatment, a fluorescence/confocal microscope. This assay has several advantages, namely: ease of set up, the ability to test numerous angiogenic/anti-angiogenic factors simultaneously, quick network formation, ability to view live or fixed tube networks, and quantifiability. To ensure successful results and limit variability, proper selection of basement membrane extracts and endothelial cells is necessary, and conditions must be optimized. In summary, this assay is a useful method for screening potential angiogenic/anti-angiogenic factors as well as identifying critical mechanisms and signaling pathways underlying angiogenic-related pathologies.