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. 2021 Dec 17;11:24151. doi: 10.1038/s41598-021-03015-2

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© The Author(s) 2021

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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(A) Side scattering intensity measured (symbols) by a CytoFLEX¹ and calculated (lines) by Mie theory¹⁰ versus diameter for polystyrene beads (squares), silica beads (circles), viruses (triangles), and CD61 + EVs (hexagons). The assumed refractive index (RI) is 1.627 for polystyrene beads, 1.440 for silica beads, and 1.472 for viruses at the illumination wavelength of 405 nm. EVs were modelled as core–shell particles, having a 12 nm thick shell with an RI of 1.52 and a core RI of 1.353 (upper solid line), and having a 4 nm thick shell with an RI of 1.450 and a core RI of 1.380 (lower solid line). The horizontal line indicates the trigger threshold. (B) Solid particle equivalent RI versus diameter for plasma-derived EVs based on Flow-SR¹¹ and literature (panel A, solid green lines), for liposomes¹⁹ (dotted line), and for plasma-derived EVs based on the dynamic light scattering (DLS) measurements and the flow cytometry light scattering measurements performed by Brittain et al.¹ (overlapping blue lines and symbols). Below a diameter of 100 nm, the solid particle equivalent RI based on DLS and flow cytometry light scattering is substantially higher compared to estimates based on Flow-SR¹¹ and literature and liposome measurements.