Exosome characterization

For Western blot analysis, proteins were extracted from isolated exosomes with Radio-Immunoprecipitation Assay (RIPA) Buffer (Sigma-Aldrich) and volumes, corresponding to 20 μg of proteins, were separated on 10% a SDS-PAGE gel. Samples were then transferred onto a nitrocellulose membrane (Bio-Rad laboratories, Hercules, CA, USA), which was subsequently blocked in 5% Non-Fat Dry Milk (Bio-Rad Laboratories). The membrane was incubated with primary antibodies against Calnexin (1:1000; clone H-70; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD9 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and CD81 (1:800; clone H-121; Santa Cruz Biotechnology, CA, USA) overnight at 4°C. Secondary antibody was ECL anti-rabbit IgG horseradish peroxidase-linked F(ab’)₂ fragment (donkey-anti rabbit, 1:10000; GE Healthcare, UK). The membrane was visualized with ECL Prime Western Blotting Detection (GE Healthcare Life Sciences) and a VersaDoc 4000 MP (Bio-Rad Laboratories).

Size determination of exosomes was assayed using Zeta View (a Nanoparticle Tracking Analysis device: Particle Matrix, Germany).

Exosomes (10μg) re-suspended in PBS were loaded onto Formvar/Carbon-coated grids (Ted Pella Inc., Redding, CA, USA) fixed in 2.5% glutaraldehyde, contrasted in 2% uranyl acetate and visualized with LEO 912AB Omega electron microscope (Carl Zeiss NTS, Jena, Germany) [39].