(A) Theoretical calculation of the number of EVs immobilised per bead. The graph represents at real scale a 6 μm-diameter bead and an EV of 150 nm of diameter (ɸ). The external surface of a bead (Sbead) could capture a maximum of N times the circle area (Cexo) of 150 nm EVs. N is calculated by dividing Sbead/Cexo. (B) Schematic representation, not to scale, of the bead binding assay. 6 μm-diameter fluorescent magnetic beads were coated with capture antibody and used for immune-capture of the EVs. A second antibody directed against the same or a different molecule, either biotinylated or directly conjugated with PE, was used for detection. In the case of biotinylated antibodies, PE-conjugated streptavidin was subsequently used. Samples were analysed by conventional flow cytometry.