Figure 3
Specificity of EV immunocapture on antibody-coated microbeads. (A) Gating strategy. EVs immobilised on 6 μm APC-beads were stained using biotinylated antibody followed by PE-conjugated streptavidin and analysed by flow cytometry. A gate containing only single beads was created in the Forward Scatter (FSC)/Side Scatter (SSC) plot. A second gate, within single beads, confirmed the APC fluorescence of microbeads. 1500–2000 events from this combined gating were acquired and analysed for PE labelling. (B) Negative control, IgG1. 109 particles of PC3-derived EVs were captured onto either anti-CD63 (Clone TEA3/18) or IgG1-coated beads followed by detection with biotinylated antibody directed against CD9. A sample with no EVs is also shown for comparison. (C) Antibody blocking. 109 particles of PC3-derived EVs were pre-incubated with increasing amounts of the indicated soluble blocking antibody [anti-CD63 (Clone TEA3/18), anti-CD9 (Clone VJ1/20)] before being incubated for capture on CD63- (left) or CD9- (right) coated beads. Experiments are representative of 3 independent repetitions.
