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Flow Cytometric Analysis of Extracellular Vesicles

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

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.

Key words

Flow cytometry Extracellular vesicles Exosomes Subsets 

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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

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