Extracellular Vesicles Isolation and Characterization

EVs were obtained from cell supernatants by ultracentrifugation as previously described [3 (link)]. In brief, CD105⁺ CSCs and CD105^- TCs were cultured overnight in RPMI (Euroclone). Cell supernatant was centrifuged twice at 3000 g for 10 min to remove cell debris and then ultracentrifuged at 100.000 g (Beckman Coulter Optima L-90 K ultracentrifuge, Brea, CA, USA) for 2 h at 4 °C. EVs were stored in serum free RPMI supplemented with 10 % DMSO at -80 °C. EV number was quantified by NanoSight LM10 instrument (NanoSight Ltd., Amesbury, UK) equipped with the nanoparticle tracking analysis (NTA) 2.0 analytic software. The protein content of EV preparations was quantified by Quantifluor (Promega) using NanoOrange Protein Quantitation Kit (Life Technologies, Carlsbad, CA). Cytofluorimetric analysis was performed by Guava easyCyte Flow Cytometer (Millipore, Billerica, MA, USA) and analyzed with InCyte software using the following FITC- or PE- conjugated antibodies: CD44, CD105, α5 integrin, α6 integrin (Miltenyi Biotec, Bergisch Gladbach, Germany), CD73, CD29, CD90 and CD146 (BD Biosciences) [48 (link)]. FITC or PE mouse isotypic IgG (Miltenyi Biotec) were used as control (Additional 3: Figure S1).