Matrigel angiogenesis assay

To study angiogenesis, 150 μl of Matrigel substrate (Corning, Corning, New York) diluted with DMEM (1:10) was pipetted into each well of a 24-well plate and allowed to solidify for 1 h at 37°C. Thereafter, 2 × 10⁴ HUVECs were seeded into each well in either endothelial GM (see details earlier in the text), UM (serum-free), CM, or ICM. Cells were imaged at 24 h using a light microscope (Olympus). Five fields of view at ×20 magnification were imaged for each condition. Parameters of angiogenesis (capillary area, number of tubes per field of view, tube length, and branching points) were analyzed using Wimasis image analysis software (WimTube Release 4.0, Onimagin Technologies, Córdoba, Spain).