Optimized protocol for isolation of small extracellular vesicles from human and murine lymphoid tissues
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 microenvi...
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| Main Authors: | , , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
4 August 2020
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| In: |
International journal of molecular sciences
Year: 2020, Volume: 21, Issue: 15 |
| ISSN: | 1422-0067 |
| DOI: | 10.3390/ijms21155586 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.3390/ijms21155586 Verlag, lizenzpflichtig, Volltext: https://www.mdpi.com/1422-0067/21/15/5586 
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| Author Notes: | Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K. Maaß and Martina Seiffert |
| Summary: | 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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| Item Description: | Gesehen am 17.09.2020 |
| Physical Description: | Online Resource |
| ISSN: | 1422-0067 |
| DOI: | 10.3390/ijms21155586 |