| Summary: | <i>Leishmania</i> parasites are a group of kinetoplastid pathogens that cause a variety of clinical disorders while maintaining cell communication by secreting extracellular vesicles. Emerging technologies have been adapted for the study of <i>Leishmania</i>-host cell interactions, to enable the broad-scale analysis of the extracellular vesicles of this parasite. <i>Leishmania</i> extracellular vesicles (<i>L</i>EVs) are spheroidal nanoparticles of polydispersed suspensions surrounded by a layer of lipid membrane. Although <i>L</i>EVs have attracted increasing attention from researchers, many aspects of their biology remain unclear, including their bioavailability and function in the complex molecular mechanisms of pathogenesis. Given the importance of <i>L</i>EVs in the parasite-host interaction, and in the parasite-parasite relationships that have emerged during the evolutionary history of these organisms, the present review provides an overview of the available data on <i>Leishmania</i>, and formulates guidelines for <i>L</i>EV research. We conclude by reporting direct methods for the isolation of specific <i>L</i>EVs from the culture supernatant of the promastigotes and amastigotes that are suitable for a range of different downstream applications, which increases the compatibility and reproducibility of the approach for the establishment of optimal and comparable isolation conditions and the complete characterization of the <i>L</i>EV, as well as the critical immunomodulatory events triggered by this important group of parasites.
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