Endothelial Cell Migration Assay

Migration of ECs toward a gradient of growth factors (EGM-2MV Microvascular Endothelial Cell Growth Medium-2, Lonza, Catalog #CC-4147) was performed in a 24-well Transwell Boyden Chamber (8.0 mm pore size, polycarbonate membrane, Corning Costar; Corning Incorporated Life Sciences, Acton, MA) as described previously (25 (link)). Briefly, 750 μl EBM-2 MV growth medium (Lonza, Catalog #CC-3202) with 10% exosome-depleted FBS were added to the lower compartment. ECs (1 × 10⁵ cells), pre-treated with exosomes, were added to the upper compartment (insert) in 200 μl of Opti-MEM (Thermo Fisher Scientific). After incubation at 37°C for 12–16 h, the cells that migrated through the membrane were stained with Giemsa stain (Sigma) and counted manually under phase contrast microscopy (Nikon Eclipse Inverted Phase Contrast Microscope, Spectra Services, Inc.; x100 magnification) in each well. Data from at least five biological replicates are expressed as the mean number of cells per well that migrated through the membrane.