Characterization of EV.

Human MSC were cultured on ibidi μ-slides, fixed and analyzed using electron microscopy. Shown is a part of cell membrane of a hMSC releasing EV (a). After ultracentrifugation of the supernatant, EV were resuspended in small volumes, sucked into carbo-tubes, fixed and analyzed using electron microscopy. Patches (b) and single EV (c) of 50–1,000 nm were detected. Ten μg of characteristic EV-proteins (CD81, HSP70, CD9 and CD63, GAPDH as housekeeper) were analyzed by Western blot (d). For quantification of EV using flow cytometry, size beads ranging from 0.2–2 μm were used to define the EV analysis area P1 (e) and impurities of 0.1μm filtered PBS in P1 (f). Purified EV in P1 were quantified using counting beads excluding the particles contained in filtered PBS. Total EV amounts per harvest (samples A-N from three individual donors) blotted against the protein content of each EV harvest revealed interindividual differences in protein cargo but reproducibility within one donor culture after repeated EV harvests (g). To investigate the underestimation of EV due to “swarm detection” in flow cytometry, 6 EV harvests were measured with NanoSight revealing ca. 1,000 fold higher concentration (401 ± 290) with a mode size of 146 ± 7.7 nm (h).