Quantifying EPC Migration Dynamics

Migration of late EPCs was measured using a modified Boyden chamber assay, as previously described (19 (link)). Briefly, following pretreatment with 20 µmol/l RES for 12 h, EPCs were cultured with TNF-α (10 ng/ml) for another 24 h in a serum-free EGM2 medium. A total of 4×10⁴ EPCs were added in the upper insert of the chamber in an EGM2 medium (Lonza Group, Ltd.,) and also EGM2 medium with fetal bovine serum was added to the bottom chamber. Following incubation for 8 h at 37°C in an incubator with 5% CO₂, the membrane in the upper chamber was washed gently with PBS and non-migratory cells were scraped using cotton swabs. Subsequently, EPCs were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with DAPI at room temperature for 15 min, and the number of migratory cells in the bottom chamber in six randomly selected fields/well was calculated under a fluorescent microscope (Leica Microsystems GmbH, Wetzlar, Germany; DMI 4000; magnification, ×100).