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. 2017 Aug 30;7:9907. doi: 10.1038/s41598-017-08826-w

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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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Separation of nanometer- and/or micrometer- biological vesicles from cell culture. (a) Separation procedures. After mild centrifugation, cells are discarded and only vesicle-suspended media is concentrated. The enriched vesicles and buffer are separately introduced to Sample and Function channels, respectively. Samples from each outlet are collected and analyzed by immunoblotting assay and TEM. (b) (c) Immunoblotting results from the separated vesicles with 75% MR are shown as cropped images from full-length western blots. Syntenin-1 and calreticulin antibodies were used as markers for 50–200 nm ranged vesicles, exosomes, and 1–5 μm of apoptotic bodies, respectively. Exosomes are clearly separated through channel #1 to #3. On the other hand, larger microvesicles including apoptotic bodies are collected outlets #5 to #9. (d) (e) Transmission electron microscopy (TEM) images of vesicles collected from different outlets: (d) exosomes at outlet #2 show a distinct cup-shape, which is a specific characteristic of exosomes. Additional vesicles sized down to 20 nm are also detected. Enlarged picture of (d) is shown in Supplementary Fig. S7. (e) Apoptotic body included aggregates from outlet #8. Round-shape vesicles (arrows at (d)) are present in outlet #2 fraction but not shown in outlet #8 fraction, indicating nanometer-sized biological particles were successfully separated from the micron particles including apoptotic bodies.