Isolation of Small Extracellular Vesicles

After selection with 4 µg/mL blasticidin-S, HuDe fibroblasts were further incubated for 72 h in serum free medium containing 4 µg/mL blasticidin-S to avoid any contamination by FBS lipoproteins. Then, medium was collected and centrifuged to remove cells, cell debris and large EVs (300× g, 10 min; 2000× g, 10 min), adding a filtration step at 0.22 µm with cellulose filter (Millipore, Burlington, MA, USA) to enrich for small EVs. Vesicles were isolated by polymer co-precipitation using Exoquick-TC precipitation method (System Biosciences, Palo Alto, CA, USA) as previously described [12 (link)]. Pelleted EVs were stored at −80 °C in PBS. EV protein content was determined by the Bradford method, using bovine serum albumin as standard.