Overview of the procedure used for isolation and characterization of secreted exosome-like vesicles from Schistosoma mansoni. (A) Adult male and female worms were cultured 48-72 h in media containing exosome-depleted serum. Vesicles were purified from the culture media by differential centrifugation, followed by filtration through a 0.2 µm membrane and ultracentrifugation on a discontinuous 25, 30, 35% sucrose gradient, as described72. The purification was monitored by western blot (WB) analysis, using antibodies against known exosomal markers (e.g. enolase) and electron microscopy (EM), prior to analyses of protein and miRNA content. (B) Schematic of the two major types of secretory extracellular vesicles. Membrane particles (or ectovesicles) are formed by outward budding of the plasma membrane. Exosomes are derived from the endocytic pathway via the formation of large multivesicular body (MVB) intermediates, which fuse with the plasma membrane releasing the vesicular cargo, exosomes, as well as other contents into an extracellular environment. Alternatively, the MVBs can be directed to lysosomes and degraded [see 24 for further details].