Table 1.

In vitro angiogenesis assays.

Type of assay	Basis	Assay	Setting	References
Proliferation (reviewed by Stoddart, 2011; Niles and Riss, 2015)	Cell number		The effect of test substance is measured by estimation of the increase in viable endothelial cell number over time	Staton et al., 2009
	Cell cycle kinetics	BrdU assay	Bromodeoxyuridine (BrdU), a pyrimidine analog, is incorporated during DNA synthesis and quantified by immunohistochemistry or ELISA	Qin et al., 2006
		Proliferation marker detection assay	Ki-67, expressed during the S, G2 and M phases, or the proliferating cell nuclear antigen (PCNA), overexpressed in the G1 and S-phase are estimated quantitatively	Whitfield et al., 2006
	Metabolism	Tetrazolium salt-assays	Metabolically active cells convert tetrazolium-salt compounds (MTT, XTT, MTS and WST1) into formazan dyes. The colorimetric change is quantified using spectrophotometry and correlated to cell number	Boncler et al., 2014
		Protease activity assay	Protease activity measured using a fluorogenic cell permeable substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC) is correlated to viable cell-number	Niles et al., 2007
		Resazurin assay:	Metabolically active cells reduce resazurin to resorufin, changing the spectrometric properties of the compound. Signal is quantified and correlated with cell number	Larson et al., 1997
		ATP-measurement	Bioluminescence-based ATP-detection assay that uses the linear relationship between viable cell number and ATP-concentration	Wang et al., 2010
	Cell death	TUNEL-assay	Fluorescent labeling of terminal deoxynucleotidyl transferase-dUTP nick end of the 3′-OH region of fragmented DNA is estimated by microscopy or flow cytometry	Goodwin, 2007
		Apoptosis marker detection assay	Expression of apoptosis cell-markers, such as caspase-3 or annexin V, is assessed via microscopy or flow cytometry	Köhler et al., 2002
		LDH assay:	The release of lactate dehyrogenase (LDH) as a consequence of loss of cell membrane integrity can be quantified to through a colorimetric reaction	Smith et al., 2011
Migration (reviewed by Hulkower and Herber, 2011)	Wound assay	Scratch assay	A tip or needle is used to remove cells to form a denuded area in a confluent endothelial cell monolayer, in which cell migration can be quantitatively estimated after a specific time interval	Steinritz et al., 2015
		Exclusion zone assay	Stencils are placed in culture plates prior to cell-seeding in order to create uniformly sized wounds in an intact confluent monolayer, in which invasion by the patterned cells can be quantitatively assessed	Gough et al., 2011
	Chemotaxis/ chemoinvasion	Boyden chamber assay	Two-compartment chamber with a semi-permeable membrane is used to evaluate active cell migration in response to specific stimuli or due to chemotaxis within a test substance gradient	Albini and Benelli, 2007
		Microfluidics assay	Creation of a diffusion-generated concentration gradient within a migration chamber, through which endothelial cells can migrate	Chung et al., 2010; Young, 2014
Morphogenesis (reviewed by Arnaoutova and Kleinman, 2010)	Tubule formation	2D-tubule formation assay	Endothelial cells are platelet on an extracellular matrix and monitored for their ability to form vessel-like tubules	Arnaoutova and Kleinman, 2010
		EC-aggregate reassembling assay	Endothelial cell spheroids or aggregates are embedded in an extracellular matrix that resembles the basement membrane environment. Upon stimulation, vessels sprout into the matrix	Li and Stuhlmann, 2011
		3D-tubule formation assay	Endothelial cells are seeded in a three-dimensional culture platform that involves extracellular matrix components and/or other cell-types. Different settings allow to study sprouting, formation, stabilization and maturation of vessel-like tubules	Hetheridge et al., 2011; Diaz-Santana et al., 2015