Exosome Isolation and Characterization

Exosomes were enriched and depleted from conditioned medium by ultracentrifugation. Medium was centrifuged at 4 °C for 30 min at 2,500×g (Thermo Fisher, Heraeus Multifuge 3SR+). The supernatant was centrifuged at 4 °C for 35 min at 4,565×g, passed through a 0.2 μm filter, and centrifuged at 4 °C for 2 h at 110,000×g in an Optima XPN ultracentrifuge with a SW32 Ti rotor (Beckman Coulter). The cleared supernatant was the exosome-depleted fraction; pellets were resuspended in Cor.4U medium as the exosome-enriched fraction. For exosome identification, 75,000 4 μm aldehyde/sulphate latex beads (Thermo Fisher) were resuspended in 0.025 mol/l MES buffer [2-(N-Morpholino)ethanesulfonic acid; Sigma-Aldrich], coated with 8 μg CD63 (Biolegend) or rat IgG2α (Biolegend), incubated on a rotator for 20 min in a final volume of 50 μl PBS, then incubated for 30 min with 100 mmol/l glycine (Sigma-Aldrich) to occupy unreacted sites. Media were incubated with the antibody-coated beads in a final volume of 250 μl for 15 min. Exosome-bead complexes were stained with 10 μg/ml FITC labelled anti-CD9 (Biolegend), with gentle agitation for 30 min in the dark. Results were visualised using an LSRII flow cytometer (Becton Dickinson) equipped with five lasers^{5 (link)}, and were analysed using FlowJo (v10, FlowJo).