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. 2017 May 12;7:1878. doi: 10.1038/s41598-017-01731-2

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© The Author(s) 2017

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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Submicron particle detection by nanoFACS HR-FCM. (A) Representative dot plot of PBS or 100, 200 and 500 nm polystyrene beads, analyzed by light scattering and fluorescence by nanoFACS, with background reference noise shown in the lower left corner in each plot. The background reference noise is a random sampling of scattered light from laser:stream intercept. (B) Size distribution of 100 and 200 nm polystyrene beads and (C) DC2.4-derived EVs (left) or control sample from EV-depleted medium subjected to same isolation procedure (right) by NTA. (D) DC2.4 EV detection by light scattering in nanoFACS, clearly resolved above the background reference noise. (E) Serially diluted DC2.4 EV analisis by nanoFACS to assess the suitable operational range that avoids coincident detection of particles. The relative percentage of noise and EVs particles changes as the EV concentration increases, but the light scattering pattern doesn’t change. (F) Quantification of the total event rate in E. Dotted line depicts the limit of the operational range. The curve fit was calculated by nonlinear regression excluding the three most concentrated EV preparations. (G) Noise rate is stable in the operational range, but drops when sample concentration is above the operational range (gate strategy shown in E). Representative data from multiple independent experiments with similar results. NTA histograms represent the mean of three independent acquisitions ± SD in green. The numbers on the NTA graphs indicate the mode value of the size. EV, extracellular vesicle; Noise, background reference noise; FSC, forward and SSC, side light scatter; NTA, Nanoparticle Tracking Analyses; SD, standard deviation.