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. 2019 Nov 5;9:16039. doi: 10.1038/s41598-019-52366-4

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© The Author(s) 2019

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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Tetraspanin Expression on the CD61⁺ plasma EVs. (A) CD61⁺ plasma EVs from human donors were tested for their expression of CD9, CD63 and CD81. The CD61⁺ EVs were around 50% CD9⁺, while CD63 and CD81 expression was mostly absent. 8.7% of the CD61⁺ EVs from Donor 2 were CD81⁺. The PBS + antibody control demonstrates the lack of fluorescent-antibody aggregates. (B) Further analysis of the CD81⁺ EVs revealed that these were predominantly CD9, CD81 and CD61 triple positive. This is reciprocally demonstrated by analyzing the CD81 expression on CD9⁺ CD61⁺ EVs, and the CD9 expression on CD81⁺ CD61⁺ EVS. In total, 7.5% of the CD61 EVs from Donor 2 were triple positive. The percentages in bold are in reference to the overall CD61⁺ EV population. These samples were serially diluted and acquired in triplicate. VSSC gain = 400; VSSC-H threshold = 3000.