Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield

Figure 2

Comparative detection of adult worm EVs isolated with three different sizes iodixanol density gradients. (a) Western blot detection of tetraspanin-2 (TSP-2) in TCA precipitated protein isolates of fractions from adult worm EVs floated bottom-up into large (12 ml), medium (4.3 ml), or small (1.7 ml) iodixanol gradients. (b) Equal 100,000 × g EV-enriched pellets of the same adult worm cultures were loaded in the three differently sized gradients. EV-containing fractions were pooled and directly mixed with sample buffer for SDS-PAGE to compare TSP-2 detection by western blot. (c) Quantification of TSP-2 band intensities of western blots in (b). (d) EVs in pooled fractions of medium and large gradients were diluted in PBS with or without 0.1%BSA, repelleted by UC, and concentrated to equal volumes as the small gradient fractions. TSP-2 levels in concentrated EV isolates from medium- and large-sized gradients were compared to EV isolates taken directly from the small density gradient. TSP-2 western blot and quantification were performed as in (b). Data in (a) and (b) is representative for 2-4 independent experiments. Each symbol in (c) and (d) corresponds to an independent experiment.

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