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. 2017 Jul 3;216(7):2217–2230. doi: 10.1083/jcb.201601109

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© 2017 Stik et al.

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Figure 1. — Characterization of AFT- and BFC-released EVs. (A–I) Electron microscopy characterization. (A and C) AFT (A) and BFC (C) cell lines showing MVB structures (arrowheads) in the cell cytoplasm. (B) MVB magnification of AFT MVBs showing round structures (arrows) of EV size. (D) Same as B, but with BFC. (E) AFT cell releasing EVs (arrows). The dotted line depicts the connection between the MVB and the cell surface. (F) AFT EV observed by cryoTEM. (G) BFC EV observed by cryoTEM. Double-headed arrows indicate the external lipid bilayer. (H and I) AFT EV (H) and BFC EV (I) observed at a larger scale. Bars: (A and C) 5 µm; (B and D) 500 nm; (E) 2 µm; (F–I) 50 nm. (J and K) Size measurement (J) and total protein quantification (K) of AFT and BFC EVs. (L) Flow cytometric analysis of CD63 (left) and CD9 (right) expression on the EV fraction from AFT and BFC cell culture medium. Box and whisker plots describe interquartile ranges and SD. Tot, total. (M) Western blot analysis of TSG101, LAMP1, CD9, and α-tubulin expression in AFT EVs and cells. (N) Bioanalyzer sizing and quantification of AFT and BFC RNA from EVs and cells. FU, fluorescent unit.