Endothelial Cell Migration Assay: Wound Healing Assay

Endothelial cell migration was studied as previously described (Alvarez-García et al., 2013a). Briefly, HUVEC control or melatonin treated for a week cells were cultured into 6-well plates (Falcon) in VCBM supplemented with 2% FBS and were allowed to reach full confluence. With a pipette tip a line of cells was scraped away. Then, cells were washed with PBS and irradiated. In the plates, four randomly selected views along the scraped line were photographed using an ORCA R2 camera (Hamamatsu Photonics, Massy Cedex, France) attached to a microscope set Nikon Ti (Werfen Group, Barcelona, Spain) at 10 × magnification. Photomicrographs were taken every 10 min during the course of the experiment, which was terminated as soon as the wound was completely filled in vehicle treated controls (after 10 h). Initial and final wound sizes were measured using the Nis Elements v.3.8 software (Nikon, Tokyo, Japan) and the difference between the two was used to determine migration distance using the following formula: initial wound size minus final wound size divided by two.