Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues
| Autor: | Sascha Dietrich, Karsten Richter, Michelle Nessling, Tobias Roider, Geraldine Genard, Marie Bordas, Kendra K Maaß, Martina Seiffert, Sibylle Ohl |
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| Rok vydání: | 2020 |
| Předmět: |
solid tissue
purification Lymphoid Tissue viruses Size-exclusion chromatography Nanoparticle tracking analysis Spleen exosomes Cell Separation small extracellular vesicles Article Catalysis lcsh:Chemistry Inorganic Chemistry ultracentrifugation Extracellular Vesicles Mice medicine Animals Humans Physical and Theoretical Chemistry lcsh:QH301-705.5 Molecular Biology Lymph node Spectroscopy Tumor microenvironment Chemistry Organic Chemistry virus diseases General Medicine lymph node respiratory system Lipids Microvesicles Computer Science Applications Cell biology size-exclusion chromatography medicine.anatomical_structure lcsh:Biology (General) lcsh:QD1-999 sucrose density cushion Cancer cell spleen Ultracentrifuge isolation |
| Zdroj: | International Journal of Molecular Sciences Volume 21 Issue 15 International Journal of Molecular Sciences, Vol 21, Iss 5586, p 5586 (2020) |
| ISSN: | 1422-0067 |
| Popis: | 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&mdash (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. |
| Databáze: | OpenAIRE |
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