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. 2020:2116:555-574.
doi: 10.1007/978-1-0716-0294-2_33.

Isolation of Extracellular Vesicles from Leishmania spp

Andrea Vucetic  1 , Alonso Da Silva Lira Filho  1 , George Dong  1 , Martin Olivier  2
Affiliations

Isolation of Extracellular Vesicles from Leishmania spp

Andrea Vucetic et al. Methods Mol Biol. 2020.

Abstract

Exosomes, a class of extracellular vesicles, are released by eukaryotes, bacteria, and archaea, as evident from both in vitro and in vivo studies. These nano-sized double-membraned vesicles play an important role in cell-to-cell communication, dysregulation of the immune system, and pathogenesis in a number of diseases, including leishmaniasis. Leishmania is a genus of obligate intracellular parasites, which infect host macrophages, are transmitted through the bite of a sandfly, and are shown to secrete exosomes with immunomodulatory activities. Given the importance of these vesicles in Leishmania spp. virulence, it is necessary to perform appropriate isolation and characterization in order to further study their relevance in the parasite's infectious life cycle. In this chapter, we describe four methods for the isolation of extracellular vesicles derived from Leishmania species including ultracentrifugation, polyethylene glycol-based precipitation, size-exclusion chromatography, and sucrose-gradient fractionation. Further, we describe the preparation of isolated samples for characterization by nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling.

Keywords: Exosomes; ExtraPEG; Extracellular vesicles; Leishmania; Micro BCA; Nanoparticle tracking analysis; Size exclusion chromatography; Sucrose gradient; Transmission electron microscopy; Trichloroacetic acid protein precipitation; Ultracentrifugation.

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References

    1. Atayde VD, Aslan H, Townsend S et al (2015) Exosome secretion by the parasitic protozoan leishmania within the sand fly midgut. Cell Rep 13(5):957–967. https://doi.org/10.1016/j.celrep.2015.09.058 - DOI - PubMed - PMC
    1. Raposo G, Nijman HW, Stoorvogel W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183(3):1161–1172 - DOI - PubMed
    1. Ogawa Y, Kanai-Azuma M, Akimoto Y et al (2008) Exosome-like vesicles with dipeptidyl peptidase IV in human saliva. Biol Pharm Bull 31(6):1059–1062. https://doi.org/10.1248/bpb.31.1059 - DOI - PubMed
    1. Trocoli Torrecilhas AC, Tonelli RR, Pavanelli WR et al (2009) Trypanosoma cruzi: parasite shed vesicles increase heart parasitism and generate an intense inflammatory response. Microbes Infect 11(1):29–39. https://doi.org/10.1016/j.micinf.2008.10.003 - DOI - PubMed
    1. Rider MA, Hurwitz SN, Meckes DG Jr (2016) ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles. Sci Rep 6:23978. https://doi.org/10.1038/srep23978 . https://www.nature.com/articles/srep23978#supplementary-information - DOI - PubMed - PMC

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