Small Extracellular Vesicle Isolation

Small EVs were isolated from cell culture supernatant by UC, as described previously [6 (link)]. Conditioned medium (240 mL) was harvested after 7 days of incubation and immediately centrifuged at 300× g for 10 min. Apoptotic bodies were removed by centrifugation at 2000× g using a Sigma13190 rotor (MBI) for 20 min. Samples were then spun at 16,500× g for 1 h using an SW28 Ti rotor (Beckman Coulter, Indianapolis, USA) to deplete microvesicles. To pellet sEVs, the same rotor was used to centrifuge samples for 3 h at 100,000× g. Collected sEVs were washed with PBS and centrifuged at 100,000× g for 1 h and finally resuspended in 200 μL PBS and stored at −80 °C. A Bradford protein assay kit (Thermo Scientific, Rockford, USA, Cat No. 23236) was used to assess sample protein concentrations. The average amount of EV protein from three replicate samples was roughly 22 ± 5, 27 ± 6 and 34 ± 6 μg for MCF10A, MCF7 and MDA-MB-231 cell lines, respectively. These samples were used for proteomic analysis.