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. 2020 Nov 4;10:19020. doi: 10.1038/s41598-020-75817-9

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Analysis of single EVs by IFCM and analysis of some of total EV population by immunoblotting. (a) Quantification, by IFCM, of EVs per mL of sample isolated from EBC1, H596 and Hs746T following EV separation by UC-DG, LSC or ExoQuick. Data is represented as mean ± S.E.M. of ≥ n = 3 repeat analysis, on samples from n = 3 biological repeats. ANOVA was used for statistical analysis; p < 0.05(*). (b) Pie-charts illustrating heterogeneity of EV populations from CM-derived EV samples from EBC1, H596 and Hs746T cells, but separated with three different methods of EV isolation. (c) Immunoblot analysis of key proteins considered to be markers of EVs, according to MISEV2018 guidelines¹⁶. Along with other accepted EV markers, included here are tetraspanins CD63 and CD9 analysed used lysates of EV heterogenous pools, which were also evaluated as single intact EVs by IFCM.