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. 2017 Nov 10;7:15297. doi: 10.1038/s41598-017-15717-7

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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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Evaluation of the isolation protocol. (a) A representative standard curve demonstrating that the CD63⁺CD81⁺ bead-coupled flow cytometry signal is proportional to the EV concentration determined by tuneable resistive pulse sensing (TRPS), allowing semi-quantitative EV measurements. (b) EV recovery after 0.22 µm filtration of cell-depleted media. The data represents flow cytometry samples A and B in Fig. 1. EV quantity in unfiltered cell-depleted media was set to 100%. (c) Protein concentration of the 24 SEC fractions determined by Bradford assay (µg/ml) and EV quantity determined by bead-coupled flow cytometry (relative fluorescent units; RFU) for the following combinations of antibodies (capture/detection): anti-CD63/anti-CD81, anti-CD63/anti-CD63, anti-CD81/anti-CD81, anti-CD9/anti-CD9 (sampling step D indicated in Fig. 1). Graph shows medians without error bars, n = 3. (d) EV mode size, mean size and concentration for SEC fractions 6–12 as determined by TRPS. The graph shows the median and range, n = 3. (e) Top: Ponceau S total protein staining of SEC fractions 5 to 18; bottom: Western blot of SEC fractions 5 to 18 for EV marker proteins CD63, CD81, MFGE8 and HSP70. For the lane MFGE8 s.p., a standardized protein concentration of 5 µg was loaded for each fraction. For all other stainings, whole fractions were precipitated and loaded on the gel without standardization of the protein content. (f) EV recovery in the final UF-SEC EV isolate (Fractions 6–11) and the final UF-SEC protein isolate (Fractions 13–19) as determined by TRPS or CD63⁺CD81⁺ flow cytometry. The EV quantity in conditioned culture medium has been set as 100%. (g) Comparison of the EV recovery for UF-SEC, UC or UC-wash, determined by TRPS (overall p-value = 0.0005) and CD63⁺CD81⁺ bead-couple flow cytometry (overall p-value = 0.0009; sampling steps E, G and H, respectively). Data was analysed using the Kruskall Wallis test followed by Dunn’s post-test. ***p < 0.001. (h) The EV-to-protein rate of purity base on TRPS and Bradford measurements of conditioned cell culture media, UF-SEC EVs and UC EVs. The statistical difference between UF-SEC EVs and UC EVs was assessed using the Mann-Whitney U test.