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. 2023 Feb 13;21:35. doi: 10.1186/s12964-023-01045-z

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© The Author(s) 2023

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — The release method enriches for subpopulation of small EVs. a Plot of size-specific events based on FITC-tagged beads (vSSC, size-related measurement). b Density and dot plots show the vSSC (size-related measurement) in function of the fluorescent signal representing positive events in samples APC-antibodies alone (b). c–e EVs simultaneously labeled with APC-tagged anti-CD81, -CD9, and/or CD63 antibodies in the absence (c) or presence of NP40 detergent (d) and overlayed EVs isolated from the conditioned media of cpVenus-HEK cells by ultracentrifugation (e). f Overlay of EVs individually labeled with the three APC-tagged antibodies and antibodies controls. g Representative overlay of PE vs. APC fluorescence signal of individually (PE-CD81, yellow; APC-CD9, red) or simultaneously (green) labeled EVs of the release method, as well as the swarming effect (turquoise). h Total number of events of simultaneously labeled EVs as a function of PE or APC fluorescence, respectively (turquoise gate: background; yellow gate: PE⁺ events; red gate: APC⁺ events). Relative abundance of gated events as a function of PE or APC fluorescence within total events (grey). i ExoView analysis of the number of positive particles for CD81 or CD9 (N = 3 samples)