Extracellular Vesicle Isolation and Characterization

Cells were cultured in EV-depleted medium, prepared by ultracentrifugation of 50% FBS (120,000g, 16 h) (Théry et al, 2006 (link); Soares et al, 2015 (link)). Conditioned medium was subjected to differential centrifugation at 4°C (10 min, 300g; 20 min, 16,500g). Supernatants were filtered (0.22 μm) and ultracentrifuged (70 min, 120,000g). Unless stated otherwise, WB analysis were performed in EVs obtained from 12 × 10⁶ secreting cells, during the indicated conditioning times and treatments. Intracardiac EVs were isolated after tissue mincing in 0.9% NaCl and centrifugation (5 min, 400g; 15 min, 2,000g; and 45 min, 16,500g). Supernatants were incubated with Total Exosome Isolation Reagent (from serum; Thermo Fisher Scientific) overnight at 4°C and centrifuged (60 min, 10,000g; 70 min, 120,000g). Blood from humans and mice was collected into non-heparinized tubes (BD Vacutainer SST II Plus plastic serum tube; BD Biosciences), allowed to cloth for 30 min (room temperature) before serum retrieval by centrifugation (15 min, 1,000g). Samples were centrifuged (30 min, 2,000g) and EVs isolated with Total Exosome Isolation Reagent (from serum), according to the manufacturer. EVs were kept at −80°C until further analysis.