Mab b11

For electron microscopy, purified Exo and MV from 0.1 ml of plasma were processed for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as previously described (Shively and Miller, 2009 (link); Paolino et al., 2021b (link)).
For the immunoelectron microscopy, the samples on the grids were incubated with anti-CD81 monoclonal antibody (mAb B11, Santa Cruz Biotechnology, Heidelberg, Germany), and with 10 nm gold-conjugated goat anti-mouse immunoglobulin G (IgG) serum (Sigma-Aldrich, St. Louis, MO). Finally, nanovesicles were observed with a PHILIPS EM208S transmission electron microscope (FEI-ThermoFisher) (Federici et al., 2020 (link)).

To investigate a homogeneous EV population and exclude lipoprotein contamination, we captured sEV expressing CD81 pan EV marker from plasma, which was depleted from large EV, i.e., after the centrifugation at 12,000× g for 45 min (see Section 2.2). Briefly, Protein A + G Sepharose resin (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) was washed twice with sodium tetraborate 0.1 M pH 9.0 and incubated with monoclonal anti-CD81 antibody (mAb B-11, Santa Cruz Biotechnology, Heidelberg, Germany) under rotation at RT. After washes, the mAb-CD81-resin was resuspended in 0.1 M sodium tetraborate containing 20 mM dimethyl pimelimidate DMP (Pierce) crosslinker and incubated o.n. To capture CD81sEV, after washing twice with 50 mM Tris pH 7.5, the mAb-CD81-resin was incubated o.n. with plasma.