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. 2021 Jul 15;41(9):2454–2468. doi: 10.1161/ATVBAHA.121.316539

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© 2021 The Authors.

Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited.

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Figure 4. — Validation of endogenous cardiovascular extracellular vesicles (EVs) from adult zebrafish.A, Schematic describing the centrifugation steps taken to isolate EV fractions following cell dissociation of adult zebrafish ventricular tissue. B and C, Cryo-EM micrograph of isolated EVs from a pool of ventricles (n=60). C, A panel of 4 higher magnification views of the boxed regions in B. D, Histogram of the size distribution of EVs visualized by cryo-EM. n refers to number of EVs analyzed. E, Typical flow cytometry scatter plots showing the gates used to sort adult cardiac EVs and the controls used to define these gates. F and G, Gaussian distribution of NTA analysis on unsorted EV fractions before and after detergent treatment (F) and after sorting for calcein+ EVs and mCherry+ calcein+ EVs (G). H, Dot blot analysis of protein extracted from FAVS isolated particles, sorted for calcein+ EVs from both nontransgenic and Tg(myl7:HRAS-mCherry) adult ventricular tissue, confirms expression of the EV components Alix and Syntenin. I, TEM-negative stain micrographs of FAVS isolated particles, sorted for mCherry+ calcein+ EVs from Tg(myl7:HRAS-mCherry) adult ventricular tissue. J, Schematic overlay describing the position of the 3 vessels visible in the integrated time series of live imaging of endothelial cell-EVs in the peripheral circulation of an adult Tg(actb2:HRAS-EGFP); Tg(kdrl:mCherry-CAAX) double transgenic fish. White and yellow arrows indicate 2 EC-EVs moving with the blood flow. Scale bars: B, 200 nm; C and I, 50 nm; J, 10 μm.