Precooled matrigel

To assess the effect of genes on angiogenesis, we performed a tube formation assay by adding 25 µL pre-cooled Matrigel (Corning) diluted in DMEM at 1:1 to 96-well plates and placed at 37 °C for 30 min. Cells were added to 96-well plates at 2 × 10⁴ cells/well and incubated for 6 h, followed by the observation of tube formation in the Matrigel under a Nikon Eclipse Ti inverted microscope (Nikon). The angiogenesis was evaluated on the basis of vessels area and total vessel length using the AngioTool, a computational tool for quantitative analysis of vascular networks [13 (link)].

Precooled Matrigel (Corning, USA) was added 24-well plates and polymerized for 30 min at 37°C. HUVECs (2 × 10⁴) suspended in 200 μl of medium from each group were added to each well and incubated at 37°C for 6–12 h. The capillary-like structures were acquired under a microscope (Nikon, Tokyo, Japan). Angiogenic activity was quantified by measuring the mesh and length of the completed tubes.

Precooled Matrigel (Corning) was added into wells of a 24-well plate and polymerized for 30 min at 37°C. Human umbilical vein endothelial cells (HUVECs) (2 × 10⁴) suspended in 200 μL of conditional medium were added to each well and incubated with the supernatants from the indicated K1 and B-CPAP cells at 37°C in 5% CO₂ for 6–12 h. The images of tube structure were captured under a 100× bright-field microscope, and quantification of tube formation was measured by the mesh and length of the completed tubes by Image View 3.7 (Jingtong, China). The experiments were repeated three times.