2.3. Quantitative ImageJ Analysis for Angiogenic Assay

The inherent ability of endothelial cells to form anastomotic interconnected tubules was quantified by measuring the attributes of the network formed. By using software ImageJ and the Angiogenesis Analyzer plug-in tool, several indicators of tube-forming elements were analyzed, including number of Junctions, Segments, Branches, Meshes, Master Junctions, Master Segments and Total Branching Length, as previously described [24].

ImageJ plug-in Angiogenesis Analyzer allowed a quantitative evaluation of the vessel-like network organization. To better understand various indicators of in vitro pseudo capillary structure, the terminology of different parameters with detailed explanation is presented as follows, based on the provided instruction [24].

A segment of angiogenic tube with a free-ending on one side, and the other end connected to a junction point, hereby forms a structure resembling a “Branch”. “Segments” represent portions of an angiogenic capillary with two ends connected to two junction points. If these two junctions are “Master Junctions”, this segment is called a “Master Segment”. “Junctions” indicate the multi-intersection junctions with three or more furcated branches in the angiogenic structures. If furcated branches of the junction are exclusively connected with other capillary structures and thereby without extremities, this junction is called a “Master Junction”. “Meshes” are closed areas formed by segments. “Total branching length” is the total length of all recognizable capillary tubes in the analyzed area, including all the segments and branches. “Total branching length” and “Total Segment Length” were analyzed and calculated.