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. 2017 Nov 10;7:15297. doi: 10.1038/s41598-017-15717-7

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© The Author(s) 2017

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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Flow chart of the EV isolation protocols. Red arrows indicate where samples were taken for determination of EV recovery based on CD63+ CD81+ bead-coupled flow cytometry. For nano LC/MS-MS, the isolation protocols were scaled up to 120 ml of cell culture medium as starting material. For UF-SEC, the 120 ml were concentrated by 10 kDa filtration and subsequently run over the SEC column in two aliquots of 0.5 ml. The EV containing fractions from both SEC runs (fractions 6 to 10 or 11) were then pooled and concentrated by 10 kDa filtration. For UC, the 120 ml were processed in 3 portions of 40 ml, corresponding to the maximal volume of the UC tubes.