Quantification of Endothelial Cell Migration

Migration was quantitated with a scratch wound assay, as previously described [12 (link)]. In detail, wounds were set by scraping cell monolayers with a sterile disposable rubber policeman. For that purpose, ECs were cultivated on 6 cm dishes, which were labeled with a trace line before setting the wound. After applying them to the injury, non-attached cells were removed by gentle washing with culture medium. Quantification of EC migration from the edge of the injured monolayer was performed by staining the cells with 500 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) (Carl Roth, Karlsruhe, Germany) in PBS after the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Microscopic pictures were taken using an Axio Observer Z (Zeiss, Jena, Germany) using a 200× magnification. The cells which had invaded the wound from the trace line were automatically counted using the particle analysis feature of Image J 1.52a after watershed separation of overlapping nuclei.