Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues
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Le résumé fourni par la source
Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols.
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Le contrôle bibliographique ouvert
DOI retrouvé dans Crossref DOI retrouvé ; titre concordant.
- Titre Crossref
- Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues
- Date Crossref
- 04/08/2020
- Éditeur
- MDPI AG
- Type
- journal-article
Ce recoupement confirme des métadonnées liées au DOI. Il ne confirme ni la méthode ni les conclusions de l’étude, et il ne compte pas comme une seconde source scientifique indépendante.
Où se fait cette recherche
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German Cancer Research Center Division of Molecular Genetics pays non établi dans la noticeStructure de recherche
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Heidelberg University pays non établi dans la noticeUniversité ou école supérieure
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DKFZ-ZMBH Alliance pays non établi dans la noticeStructure de recherche
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Hopp Children's Cancer Center Heidelberg pays non établi dans la noticeStructure de recherche
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Faculty of Biosciences pays non établi dans la noticeUniversité ou école supérieure
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Central Unit Electron Microscopy pays non établi dans la noticeInstitution
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University of Heidelberg Department of Medicine V pays non établi dans la noticeUniversité ou école supérieure
Division of Molecular Genetics — German Cancer Research Center, Heidelberg University et DKFZ-ZMBH Alliance, avec 4 autres affiliations.
Une affiliation ne permet pas de déduire la nationalité d’un auteur.