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. 2018 Mar 16;8:4662. doi: 10.1038/s41598-018-23112-z

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© The Author(s) 2018

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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Blocking the exofacial phosphatidylserine binding sites of embryo-derived EVs inhibits the binding of EVs to the lymphocytes. Prior to incubating embryo-derived EVs with anti-CD4 and anti-CD8 labelled splenocytes, we masked phosphatidylserine on the membranes of EVs by AnnexinV, or the phosphatidylserine receptors on the lymphocytes and checked the number of EVs binding CD4+ and CD8+ cells. Panel A: The upper right quadrants of representative dot plots show the PKH+/CD4+ or PKH+ /CD8+ double positive cells. These populations represent the number of lymphocytes which bound PKH-labelled EVs on their surface. Both anti-PSR antibody pretreatment of lymphocytes, and blocking of the exofacial phosphatidylserine on embryo-derived EVs by Annexin V partially inhibited the binding of EVs. Panel B: For comparing EV binding of lymphocytes with or without blocking, we used the PKH fluorescence intensity parameter (geometric mean channel values/MFI values) which reflects on the number of EVs per cell. PKH fluorescence was measured in the lymphocyte gate. Significantly lower (p = 0.05) PKH fluorescence was detected both after anti-PSR antibody pretreatment and Annexin V blocking. The bars represent the mean+/− SEM of 4 experiments.