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. 2021 Jul 27;3(3):428–440. doi: 10.1016/j.jaccao.2021.05.006

Figure 2.

Figure 2

EV Size and Mitochondrial Content Characterization

(A, top left) Representative dot plot of MSC-EVs displaying a population of MitoTracker Green positive [MTG(+)] and MitoTracker Deep Red positive [MTDR(+)] EVs in the L-EV (>200 nm) size range by side scatter. (A, top right) Dot plot of the same MSC-EVs in top left gated to show MTG(+)/MTDR(+) vesicles (red oval). (A, bottom) Apogee bead reference dot plot (right) and size histogram (left). (B) Representative dot plot for L-EVs showing a distinct MTG(+)/MTDR(+) population (red oval). (C, top) Representative dot plot for L-EVs with accompanying qNano analysis demonstrated a mean particle size of 352 nm (216-498 nm). (C, middle) Representative dot plot for S-EVs showing smaller side scatter profile lacking MTG signal with accompanying qNano analysis showing mean particle size of 92 nm (58-122 nm). (C, bottom) Representative dot plot for BD FACSAria sorting of MSC-EVs showing relative size by side scatter of mitochondria(+) EVs (red) vs mitochondria(−) EVs (green) with accompanying qNano analysis of sorted mitochondria(+) EVs showing mean particle size of 428 nm (350-800 nm), similar to L-EVs. (D, top) TEM image of L-EVs measuring 250-500 nm. (D, bottom) Immunogold (10 nm) labeling showing 200-500 nm L-EVs are CD9(+) (red arrows) (n = 3). (E) Western blot confirming presence of complete mitochondria within L-EVs and MSC-lysate represented by outer membrane (VDAC), inner membrane (COX4), and matrix (pyruvate dehydrogenase) proteins (n = 3). COX4 = cytochrome C oxidase subunit 4; VDAC = voltage dependent anion channel; other abbreviations as in Figure 3.