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. 2019 Feb 12;9:1837. doi: 10.1038/s41598-018-38320-w

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© The Author(s) 2019

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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Comparison of AFSC-EV protein content and EV markers. (a) Protein quantification of AFSC-EV preparations using the Pierce Bradford assay. Data are shown as mean ± SD n = 3. No difference was found between preparations isolated with UC, ExoQuick and TEIR. AFSC-EV preparations isolated using qEV had lower protein content than those isolated using Exo-PREP (p < 0.05). (b) Correlation analysis between total number of particles analyzed with Nanoparticle Tracking Analysis (Fig. 1c) and EV protein concentration (μg/μL) in each preparation of AFSC-EVs obtained by different isolation techniques [p = 0.25, r = 0.6 (95% CI −0.56 to 0.97)]. (c) Expression of canonical EV markers Hsp70, CD63, Flotillin-1, and TSG101 obtained by Western blot analysis for the different isolation techniques. All AFSC-EV isolation techniques showed no evidence of residual cellular debris, as evidenced by a lack of H3K27me3 protein expression. AFSCs (parent cells) and AFSC-conditioned medium (AFSC-CM; the initial starting material from which all techniques were derived), are shown as positive controls. Representative photo from n = 3 replicate analyses.