Flow Cytometric Analysis of Extracellular Vesicles

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.

Keywords: Exosomes; Extracellular vesicles; Flow cytometry; Subsets.

Fig. 1

Relative sizes of EVs, cells, and antibodies. The typical size of a laser intercept at the point of flow cytometric analysis of cells, beads, or EVs is 10–20 μm, while exosomes and similar EVs are ~0.1 μm. The objects in this figure are drawn to scale, to illustrate relative sizes of relevant structures and objects

Fig. 2

Detection of EVs and EV-associated surface molecules by binding EVs to beads. To analyze EVs with conventional flow cytometers, it is generally necessary to bind EVs to beads that are large enough to be individually resolved on the flow cytometer. The objects are not drawn to scale. Rather, they are drawn to best illustrate the conceptual assembly of the beads with ligands

Fig. 3

Flowchart for capture and analysis of EVs by binding to beads. Shown here is a general method for using streptavidin-coated beads to capture biotinylated antibodies, prior washing the beads (to remove unbound antibodies), capturing EVs, and staining the bead-bound EVs with directly conjugated antibodies

Fig. 4

Example analysis of epitope detection EVs by binding to beads. EVs isolated from DC2.4 and 4T1 cell cultures (dark grey histograms), as well as control EV-depleted medium (light grey histograms), were incubated with anti-CD9 coated magnetic beads overnight, and subsequently labeled with anti-CD9-FITC antibodies. The same clone of anti-CD9 was used for capture and detection, to ensure EV-anchored CD9 detection and not free (soluble) CD9 detection, if any. Open histograms with (asterisk) correspond to FITC-CD9 staining profile on the surface of EVs from DC2.4 and 4T1 cell lines, while the other histograms represent isotype and nonspecific binding controls