Antibody-Based Assays for Phenotyping of Extracellular Vesicles

Figure 2

Schematic view of flow cytometric phenotyping of EVs. (a) Detection of bead-associated EVs. The EVs can be absorbed onto micron-sized beads, either directly or by antibodies coated to the beads. The latter approach causes a preselection of a subtype of EVs, based on the coating antibody. Subsequently, the absorbed EVs can be phenotyped by staining with selected, fluorophores-conjugated antibodies. The instrumental setup of lasers and optics allows for the detection of the EV-associated fluorescence. Moreover, the size of the beads facilitates detection with conventional flow cytometers (LOD ~ 300 nm). The gating strategy includes the creation of a singlet gate in a forward scatter (FSC) versus side scatter (SSC) plot, thus excluding aggregates. With this analysis, the relative amount of each investigated marker can be obtained for the entire population of bead-associated EVs. (b) Detection of individual EVs. The workflow for the flow cytometric phenotyping of individual/single EVs shares similarities to that described for bead-associated EVs. However, several differences also exist. First, there is a frequent requirement for a highly pure and enriched EV preparation prior to antibody labeling, especially when analyzing exosomes. Next, dedicated flow cytometers are used, lowering the LOD to approximately 150 nm. The use of SSC (in log scale) is often used as trigger to set the threshold for detectable events. However, labeling of all EVs with a fluorescent dye, with a subsequent use of this fluorescence signal as trigger, can serve as an alternative identifier. This may aid in increasing the signal-to-noise ratio, which is highly relevant for FCM analysis of EVs. The gating strategy involves setting a predefined EV gate based on the analysis of beads with a known size (0.2, 0.5, and 0.9 μm). This both places the relevant gate and allows for a size evaluation of the EVs. The relative amount of each marker present on the EVs can subsequently be evaluated more precisely than for the bead-associated EVs, revealing the heterogeneity of EV phenotypes. Furthermore, enumeration of the EVs is possible by addition of a known amount of fluorescent beads or by analyzing an absolute volume of EV sample.

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