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Analysis of Individual Extracellular Vesicles by Flow Cytometry

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Flow Cytometry Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1678))

Abstract

Extracellular vesicles (EVs) are released by cells and can be found in cell culture supernatants and biofluids. EVs carry proteins, nucleic acids, and other cellular components and can deliver these to nearby or distant cells, making EVs of interest as both disease biomarkers and therapeutic targets. EVs in biofluids are heterogeneous, coming from different cell types and from different sources with the cell, which limits the usefulness of bulk EV analysis methods that report the average features of all EVs present. Single-particle measurements such as flow cytometry would be preferred, but the small size and low abundance of surface antigens challenges conventional flow cytometry approaches, leading to the development of vesicle-specific assays and experimental design. Among the key issues that have emerged are: (a) judicious choice of detection (triggering) approach; (b) appropriate control experiments to confirm the vesicular nature of the detected events and the contribution of coincidence (aka swarm detection); and (c) the importance of fluorescence calibration to allow data to be compared over time and between laboratories. We illustrate these issues in the context of fluorescence-triggered Vesicle Flow Cytometry (VFC), a general approach to the quantitative measurement of EV number, size, and surface marker expression.

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Acknowledgments

Supported by: UH2TR000931 from the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director.

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Authors and Affiliations

  1. Scintillon Institute, San Diego, CA, 92121, USA

    John P. Nolan & Erika Duggan

Authors
  1. John P. Nolan
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  2. Erika Duggan
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Corresponding author

Correspondence to John P. Nolan .

Editor information

Editors and Affiliations

  1. Flow Cytometry Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Teresa S. Hawley

  2. Department of Anatomy and Regenerative Biology, School of Medicine and Health Sciences, George Washington University, Washington, District of Columbia, USA

    Robert G. Hawley

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Nolan, J.P., Duggan, E. (2018). Analysis of Individual Extracellular Vesicles by Flow Cytometry. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 1678. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7346-0_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7346-0_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7344-6

  • Online ISBN: 978-1-4939-7346-0

  • eBook Packages: Springer Protocols

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