Flow Cytometric Analysis of Extracellular Vesicles

  • Aizea Morales-Kastresana
  • Jennifer C. JonesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1545)


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.

Key words

Flow cytometry Extracellular vesicles Exosomes Subsets 


  1. 1.
    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):10998–11006CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Arakelyan A et al (2013) Nanoparticle-based flow virometry for the analysis of individual virions. J Clin Invest 123(9):3716–3727CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Erdbrugger U, Lannigan J (2016) Analytical challenges of extracellular vesicle detection: a comparison of different techniques. Cytometry A 89(2):123–134CrossRefPubMedGoogle Scholar
  4. 4.
    Higginbotham JN et al (2016) Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J Extracell Vesicles 5:29254CrossRefPubMedGoogle Scholar
  5. 5.
    Danielson KM et al (2016) Diurnal variations of circulating extracellular vesicles measured by nano flow cytometry. PLoS One 11(1):e0144678CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    van der Pol E et al (2012) Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 10(5):919–930CrossRefPubMedGoogle Scholar
  7. 7.
    Lasser C, Eldh M, Lotvall J (2012) Isolation and characterization of RNA-containing exosomes. J Vis Exp 59:e3037Google Scholar
  8. 8.
    Thery C et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:22PubMedGoogle Scholar
  9. 9.
    Lobb RJ et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 4:27031CrossRefPubMedGoogle Scholar
  10. 10.
    Baumgarth N, Bigos M (2004) Optimization of emission optics for multicolor flow cytometry. Methods Cell Biol 75:3–22CrossRefPubMedGoogle Scholar
  11. 11.
    Maecker HT et al (2004) Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62(2):169–173CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.National Cancer InstituteNational Institutes of HealthBethesdaUSA
  2. 2.Molecular Immunogenetics & Vaccine Research Section Vaccine BranchCCRBethesdaUSA

Personalised recommendations