Assessing Cell Migration in Cardiomyoblasts

As previously described, a cell migration assay on cardiomyoblast cell line (H9C2) was performed [3 (link)]. Briefly, a confluent monolayer of H9C2 cells (CRL-1446, ATCC, Manassas, VA, USA) was scratched with a 200 µL pipette tip, then the cells were washed in PBS and incubated with 5 × 10⁸ EVs/mL in 1% EV-depleted (fetal bovine serum) FBS medium for 24 h, and PBS was used as negative control. The cell cultures were fixed in 4% formaldehyde and stained with Fluoromount g (DAPI, SouthernBiotech Birmingham, Al, USA). An EVOS cell imaging system (Thermo Fisher Scientific, Waltham, MA, USA) was used to take digital images of the stained cell cultures. ImageJ Freeware (imagej.nih.gov/version 1.8.0, National Institutes of Health, Bethesda, MD, USA) was used to quantify differences in the scratch areas between the experimental groups.