Figure 4

Dependence of the angiogenic activity of HUVECs on passage number. (A) Vascular networks. Cells at passages 5, 7, and 12 were encapsulated in a collagen gel, and VE-cadherin (Alexa Fluor 488) and actin (rhodamine-phalloidin) were stained at 48 hours of culture. The scale bar is 200 μm. (B) Three-dimensional configuration of vascular networks. Spatial distributions of cells at 48 hours of culture were visualized with a confocal laser-scanning microscope and were analysed using image analysis software (IMARIS). Actin (rhodamine-phalloidin) and nuclei (DAPI) were stained. (C) Capillary lengths and branching points were quantified using image analysis as shown in (B).