Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols

Abstract Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and re...

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Autores principales:	Annamaria Nigro, Annamaria Finardi, Marzia M. Ferraro, Daniela E. Manno, Angelo Quattrini, Roberto Furlan, Alessandro Romano
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2b49b54b2e4d447f9e7e417064897b70
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spelling	oai:doaj.org-article:2b49b54b2e4d447f9e7e417064897b702021-12-02T14:27:02ZSelective loss of microvesicles is a major issue of the differential centrifugation isolation protocols10.1038/s41598-021-83241-w2045-2322https://doaj.org/article/2b49b54b2e4d447f9e7e417064897b702021-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-83241-whttps://doaj.org/toc/2045-2322Abstract Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.Annamaria NigroAnnamaria FinardiMarzia M. FerraroDaniela E. MannoAngelo QuattriniRoberto FurlanAlessandro RomanoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Annamaria Nigro Annamaria Finardi Marzia M. Ferraro Daniela E. Manno Angelo Quattrini Roberto Furlan Alessandro Romano Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols
description	Abstract Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.
format	article
author	Annamaria Nigro Annamaria Finardi Marzia M. Ferraro Daniela E. Manno Angelo Quattrini Roberto Furlan Alessandro Romano
author_facet	Annamaria Nigro Annamaria Finardi Marzia M. Ferraro Daniela E. Manno Angelo Quattrini Roberto Furlan Alessandro Romano
author_sort	Annamaria Nigro
title	Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols
title_short	Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols
title_full	Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols
title_fullStr	Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols
title_full_unstemmed	Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols
title_sort	selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/2b49b54b2e4d447f9e7e417064897b70
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Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols

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