Isolation of Extracellular Vesicles by C-DGUG

EVs were isolated using Cushioned-Density Gradient Ultracentrifugation (C-DGUG) [53 (link),54 (link)]. Briefly, ~40 mL of the serum/antibiotic-free media was centrifuged at 3000× g for 15 min to eliminate cell debris, followed by filtration (0.22 µm). The filtered media were added to a liquid cushion (60% Iodixanol) and ultracentrifuged at 150,000× g for 4 h at 4 °C to concentrate the small vesicles. Subsequently, these vesicles were resolved in a density gradient (40, 20, 10, and 5% iodixanol) and submitted to ultracentrifugation at 100,000× g for 18 h at 4 °C. The EV-rich, protein-low fractions (6 and 7) were then pooled and concentrated to 500 µL on an Amicon Ultra-4 Centrifugal Filter Unit with an Ultracel-10 membrane (MWCO = 10 kDa; Merck Millipore, Darmstadt, Germany, United States Affiliate) by centrifugation at 4000× g for 30 min.