Fig 2: Vesicles secreted from cultured microvascular endothelial cells have properties consistent with identification as exosomes and contain Cx43. (A) Screen shot showing single frame of a video from the nanoparticle tracking analysis of the 100,000× g material. (B) Graph shows a representative profile of the extracellular vesicles in the 100,000× g pellet from the nano-tracking analysis. For this example, the mode was 120.7 nm; mean ± SD was 154 ± 54 nm; 10% of the vesicles were <98 nm; 50% were <141 nm; and 90% were <226 nm. (C) Immunoblots were performed on an hCMEC/D3 cell homogenate and the pellets obtained by sequential centrifugation of conditioned medium at increasing forces (300× g, 2000× g, 10,000× g and 100,000× g for the times noted in Materials and Methods). To facilitate comparisons, the gels were loaded with comparable percentages of the pellets from the sequential spins. The 100,000× g material contained the exosomal markers (flotillin-1, CD63, CD81 and Alix) as well as Cx43, but it did not contain detectable amounts of the endoplasmic reticulum protein Grp94 or VE-cadherin.

Fig 3: Western blotting for exosome identification. Representative image of Western blotting analysis of CD63 and CD81 expression on exosomes obtained by cell culture media named CTR-undiff and CTR–diff and GTP-undiff and GTP-diff samples. The image showed the presence of these proteins in the exosome samples we isolated by ultracentrifugation (see section “Materials and Methods”).

Fig 4: Characterization of EV populations by tetraspanin co-localization analysis. (a), Representative single-vesicle fluorescence images of HEK293 DUC-EVs. The CD9, CD63, and CD81 images were coloured in green, red and blue, respectively. Size of images: 17 × 17 µm. (b) Intensity line scan of selected area (yellow box). Region (i) CD9·CD63·CD81 triple-positive EV, region (ii) CD63 single-positive EVs, and region (iii) CD9·CD81 double-positive EVs. Size of enlarged images: 5 × 5 µm. (c) Pie chart of EV populations expressing different combinations tetraspanins. (d) Venn diagram depicting tetraspanin relationships in individual HEK293 EVs. The EV populations were roughly divided into two distinct groups: CD63 single-positive and CD9·CD81 double-positive EVs. (e-g) Tetraspanin co-localization analysis of MCF-7 EVs. The population of MCF-7 EVs were overall similar to HEK293 EVs. Size of images: 17 × 17 µm. (h-j) Tetraspanin co-localization analysis of B16BL6 EVs. The population of B16BL6 EVs was completely different compared to the other two cell lines; it showed higher single-positive EV proportion than the other two cases. Numbers in pie charts: mean ± S.D. (n > 8). Numbers in Venn diagrams: percentages of total EVs

Fig 5: Characterization of exosomes isolated from amniotic membrane (AM). Exosomes were isolated from conditioned media prepared from AM under normoxic (AM/N) or hypoxic (AM/H) conditions as described in the Methods. Exosomes were examined by transmission electron microscopy (A). Representative exosomes are indicated by white arrows. Scale bar = 100 nm. The presence of tetraspanins in exosomes was determined by SDS-PAGE followed by Western blot using antibodies against human CD9, CD63, and CD81 (B). The particle size of exosomes was analyzed using dynamic light scattering. The size distributions were graphed against the percentage of intensity (C).