Figure 1.

Submicron particle detection and multiparametric characterization of circulating “small EV” (sEV) by ImageStream^x (ISx). Gating sEV populations is achieved by assessment of the Scatter intensity of particles of known size, and through confirmation by single-event visual interrogation. (A) Fluorescently labeled polystyrene beads and liposomes which have a refractive index closer to that of EV are used and contrasted to CFDA-SE labeled circulating human extracellular vesicles (EVs) derived by ultracentrifugation. CFDA-SE-mediated labeling of intact vesicles is demonstrated by detergent lysis (0.1% Triton™ X-100) of UC-derived EVs prior to labeling. (B) The acquisition of appropriate control samples is an important step prior to running experimental samples if the common pitfalls of flow-cytometric profiling of EVs are to be avoided. Representative ISx dot-plots of buffer alone (filtered phosphate-buffered saline), unstained EVs, and buffer plus reagents without EVs are shown. (C) Representative dot-plot of CFDA-SE Vs. Scatter intensity to demonstrate the heterogeneity of acquired events and the principle of visual interrogation in gate-setting. The difference in properties of the two CFDA-SE-positive populations (G1 and G2) is clear by visual interrogation, showing G2 to be predominantly composed of cellular debris or particle aggregates as opposed to the uniform particles in G1—the sEV gate. (D) Multiparametric phenotyping of these gated sEV (G1) is demonstrated by fluorescent labeling with EV markers, the tetraspanins CD9, CD63, and CD81 combined (PE, Ch03) and HLA-DR (PECy7, Ch06). Fluorescence minus one and isotype controls are used to set gating. A buffer + reagent + antibody control should be performed and is demonstrated here.