FIGURE 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