FIGURE 4.

Chitosan‐mediated sEV isolation from human plasma. (a) Human plasma (0.25 ml) was diluted 1:4 with PBS and subjected to pre‐clearing as described in Figure 1, followed by sEV isolation by scUCF, the addition of the acidic formulation of chitosan to a final concentration of 50 μg/ml, or buffer. Western blot analyses of canonical EV markers CD63, CD9, HSC70, and FLOT1, as well as the non‐sEV marker CANX; total cell lysate from MCF‐10A cells was used as a positive control for CANX. (b) Western blot analyses of common contaminant plasma proteins APO‐A1 and APOB as well as FLOT1, a canonical EV marker; total cell lysate from HEK‐293 cells and 5 μl of plasma lysate (undiluted) were used as a positive controls for CANX, APO‐A1, and APOB. (c) NTA analysis of sEVs isolated from plasma by scUCF, the addition of the acidic formulation of chitosan to a final concentration of 50 μg/ml, or buffer. Particle concentration (particles/ml) is plotted against size (nm) of particles. Mean plus the standard error of the mean (n = 3) is shown