fig3

Figure 3. Characterization of sEV using NTA, western blot and TEM. (A) box plot (line at mean) of the number of particles per µg of total protein present per mL of sEV sample for the four different purification methods (*** P = 0.0008, **P = 0.0015, *P = 0.017). Three biological replicates were collected at the end of each isolation method for NTA; (B) relative abundance of exosome/EV membrane proteins (CD81, CD9, and Flot-1) and non-exosome protein marker (BSA) in sEV samples isolated from four different methodologies. Three biological replicates were pooled to create a representative sample, which was then utilized for three technical replicates (3 lanes in the Western Blot gel). Positive control is bovine plasma; (C) TEM images depict the spherical and cup-shaped sEV vesicles with appropriate size (~30-200 nm); (D) representation of original NTA images demonstrates that the EVs isolated from each method predominantly fall approximately between 30-200 nm in size.