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. 2022 Jan 20;13:405. doi: 10.1038/s41467-021-27893-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (*p = 0.038, **p = 0.0061 and **p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2⁺ EVs with CD63⁺ EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2⁻) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2⁻ (ev1Con) and ACE2⁺ (ev1ACE2) and HeLa-derived ACE2⁻ (ev2Con) and ACE2⁺ (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2⁻ and ACE2⁺) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively (N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2⁻ (evCon, left) and ACE2⁺ (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2⁺ EVs (N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2⁺ counts). Control EVs are in light blue and ACE2⁺ EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63⁺ (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively (n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2⁺ EVs.