Figure 1. Characterization of exosomes isolated by differential ultracentrifugation. (A) Western blot analysis to assess expression of exosomal markers in MRC5 exosomes. “Cells” refers to total cellular protein, “all EVs” refers to the total vesicular fraction isolated by a single ultracentrifugation of conditioned medium at 100 000 g, and “exosomes” refers to small extracellular vesicles isolated by filtration and serial centrifugation. The exosomal fraction is enriched in tetraspanins (CD63 and CD81), endosomal markers (Alix and TSG101) and does not contain Golgi (GM130) or mitochondrial (cytochrome C) markers. Actin was used as an equal loading control. (B) TEM of MRC5 fibroblast exosomes at 80 000x and 120 000x demonstrating homogenous, cup-shaped vesicles with size in the order of 100 nm. Scale bar represents 200 nm in both panels. (C) Nanoparticle tracking analysis of MRC5 fibroblast exosomes represented as size vs. concentration.