Abstract
To analyze EVs with conventional flow cytometers, most researchers will find it necessary to bind EVs to beads that are large enough to be individually resolved on the flow cytometer available in their lab or facility. Although high-resolution flow cytometers are available and are being used for EV analysis, the use of these instruments for studying EVs requires careful use and validation by experienced small-particle flow cytometrists, beyond the scope of this chapter. Shown here is a method for using streptavidin-coated beads to capture biotinylated antibodies, and stain the bead-bound EVs with directly conjugated antibodies. We find that this method is a useful tool not only on its own, without further high resolution flow cytometric analysis, but also as a means for optimizing staining methods and testing new labels for later use in high resolution, single EV flow cytometric studies. The end of the chapter includes sphere-packing calculations to quantify aspects of EV- and bead-surface geometry, as a reference for use as readers of this chapter optimize their own flow cytometry assays with EVs.
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References
Zhu S et al (2014) Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS Nano 8(10):10998–11006
Arakelyan A et al (2013) Nanoparticle-based flow virometry for the analysis of individual virions. J Clin Invest 123(9):3716–3727
Erdbrugger U, Lannigan J (2016) Analytical challenges of extracellular vesicle detection: a comparison of different techniques. Cytometry A 89(2):123–134
Higginbotham JN et al (2016) Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J Extracell Vesicles 5:29254
Danielson KM et al (2016) Diurnal variations of circulating extracellular vesicles measured by nano flow cytometry. PLoS One 11(1):e0144678
van der Pol E et al (2012) Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 10(5):919–930
Lasser C, Eldh M, Lotvall J (2012) Isolation and characterization of RNA-containing exosomes. J Vis Exp 59:e3037
Thery C et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:22
Lobb RJ et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 4:27031
Baumgarth N, Bigos M (2004) Optimization of emission optics for multicolor flow cytometry. Methods Cell Biol 75:3–22
Maecker HT et al (2004) Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62(2):169–173
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Morales-Kastresana, A., Jones, J.C. (2017). Flow Cytometric Analysis of Extracellular Vesicles. In: Hill, A. (eds) Exosomes and Microvesicles. Methods in Molecular Biology, vol 1545. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6728-5_16
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DOI: https://doi.org/10.1007/978-1-4939-6728-5_16
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6726-1
Online ISBN: 978-1-4939-6728-5
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