Jem 1010

EVs were phenotypically and structurally characterized by Transmission Electron Microscopy (TEM) with immuno-gold labelling against CD9, as previously described by Nielsen et al [44 (link)]. Briefly, 5 μl of EV isolate was mounted on a grid (SPI Supplies, PA, USA) and stained with one drop of 1% (w/v) phosphotungstic acid (pH 7.0, Ted Pella, Caspilor AB, Sweden), and subsequently blotted dry on filter paper. To visualize the presence of EV-specific marker CD9 on the surface of vesicles, IEM was performed on the isolated vesicles. The pelleted vesicles were positioned on a grid as described above and then blocked in ovalbumin. Subsequently, the grid was incubated with primary anti-CD9 antibody (1:50, BD Pharmingen, CA, USA), followed by incubation with secondary goat anti-mouse antibody conjugated with 10 nm colloidal gold (1:25, British BioCell, UK). The grids were stained with 1% (w/v) phosphotungstic acid at pH 7.0 and blotted dry. Images were obtained with a transmission electron microscopy (JEM 1010, Germany) operated at 60 keV coupled to an electron-sensitive CCD camera (KeenView, Olympus, PA, USA). Lastly, a grid-size replica (2,160 lines/mm) was imported to ImageJ 1.50i software, which enables a correct determination of the size of EVs.