Plasma Exosome Isolation and Characterization

MiniSEC was used to isolate plasma-derived exosomes and was routinely performed as detailed in our previous publications [15 (link),51 (link)]. In short, plasma samples were centrifuged at 2000 g for 10 min, at 10,000 g for 30 min, and ultrafiltrated using a 0.22 µm filter to remove impurities. One milliliter of plasma was added to a miniSEC column and eluted with phosphate-buffered saline (PBS). The fourth fraction was collected and concentrated using Amicon Ultra columns (Merck Millipore, Burlington, MA, USA). Extracellular vesicles isolated from plasma were characterized according to the MISEV 2018 guidelines for the definition of extracellular vesicles [52 (link)]. Isolation of exosomes was confirmed by assessing particle size and count using nanoparticle tracking (ZetaView PMX-220, Particle Metrix), morphology by transmission electron microscopy, and origin from the endosomal compartment by Western Blot.