Isolation and Analysis of Fasciola hepatica Extracellular Vesicles.

The finding of extracellular vesicles (EVs) as important players in parasite-parasite and host-parasite communications has led to an increasing number of reports in the literature. Different protocols have been developed for isolation and further characterization of EVs from parasitic helminths. In this chapter, we describe step by step procedures to isolate EVs secreted by Fasciola hepatica adults in culture, which could be also applied for other developmental stages of the parasite, as well as EVs present in plasma and urine. Along with classical isolation methods like differential ultracentrifugation, and more recent techniques like size exclusion chromatography (SEC), here we also refer to the storage of EVs for further functional assays. In addition, characterization of F. hepatica by electron microscopy techniques like immuno-gold staining, as well as labeling techniques useful for functional assays, like in vitro uptake of fluorescent EVs by cells in culture are also described.