2.16. In Vitro Tubule-Like Formation

The ECMatrix assay kit (Chemicon, Millipore, USA) was used to study tubule formation in vitro according to the manufacturer’s instructions. For the test, 15-well microslides (IBIDI Munich, Germany) were coated with 10 μL of Matrigel/well and left to solidify at 37 °C for 30 min. A total of 1.5 × 10⁴ HUVECs for each well were seeded and a CM (diluted 1:4) derived from ARPE-19 was added. DMEM-F12 was used as the control. The micro-slide was then incubated, under controlled conditions, for up to 12–16 h. The degree of angiogenic response was assessed by acquiring images of each well using a Nikon inverted phase contrast microscope. The quantitative analysis was obtained through Image J software [27], considering the branching index, that is, the number of junctions formed for each field. In addition, mean values and standard deviation (SD) were determined for each analysis. Three independent experiments were performed for each treatment.