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. 2020 Sep 16;10:15160. doi: 10.1038/s41598-020-72122-3

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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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Extracellular vesicles derived from genetically modified tumor cells expressing 4-1BBL or OX40L harbor respective ligands on the surface. We developed a flow cytometry assay in which EVs are captured using polystyrene beads coupled to anti-4-1BBL or anti-OX40L. The EVs bound to beads were subsequently stained for tetraspanins markers CD9 and CD81. (A) Dot plot for flow cytometry assay with 41BBL-EVs. (B) Dot plot for flow cytometry assay with OX40L-EVs. For all dot plots y-axis is the SSC, and x-axis is FITC intensity for CD9-FITC or APC intensity for CD81-APC. (C) Graphic representation of positive EVs for 4-1BBL-CD9, 4-1BBL-CD81, OX40L-CD9 and OX40L-CD81. The bars represent the mean ± SD of three independent experiments. The T test was performed (**p < 0.005; ***p < 0.0001). (D) Flow cytometry histogram shows 41BB-EVs and OX40L-EVs. The y-axis represents the percentage of cell numbers and the x-axis is the mean fluorescence intensity (MFI) on a logarithmic scale. Red curves represent parental-EVs used as a control, green curves are EVs preparations positives for CD9 and blue curves are EVs preparations positive for CD81. The figures are representative of three independent experiments.