Labeling Extracellular Vesicles for Nanoscale Flow Cytometry

Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, are 30–800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and...

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Autores principales:	Aizea Morales-Kastresana, Bill Telford, Thomas A. Musich, Katherine McKinnon, Cassandra Clayborne, Zach Braig, Ari Rosner, Thorsten Demberg, Dionysios C. Watson, Tatiana S. Karpova, Gordon J. Freeman, Rosemarie H. DeKruyff, George N. Pavlakis, Masaki Terabe, Marjorie Robert-Guroff, Jay A. Berzofsky, Jennifer C. Jones
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fc12a330525a480593a5dd57e9326f33
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spelling	oai:doaj.org-article:fc12a330525a480593a5dd57e9326f332021-12-02T12:32:51ZLabeling Extracellular Vesicles for Nanoscale Flow Cytometry10.1038/s41598-017-01731-22045-2322https://doaj.org/article/fc12a330525a480593a5dd57e9326f332017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01731-2https://doaj.org/toc/2045-2322Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, are 30–800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.Aizea Morales-KastresanaBill TelfordThomas A. MusichKatherine McKinnonCassandra ClayborneZach BraigAri RosnerThorsten DembergDionysios C. WatsonTatiana S. KarpovaGordon J. FreemanRosemarie H. DeKruyffGeorge N. PavlakisMasaki TerabeMarjorie Robert-GuroffJay A. BerzofskyJennifer C. JonesNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Aizea Morales-Kastresana Bill Telford Thomas A. Musich Katherine McKinnon Cassandra Clayborne Zach Braig Ari Rosner Thorsten Demberg Dionysios C. Watson Tatiana S. Karpova Gordon J. Freeman Rosemarie H. DeKruyff George N. Pavlakis Masaki Terabe Marjorie Robert-Guroff Jay A. Berzofsky Jennifer C. Jones Labeling Extracellular Vesicles for Nanoscale Flow Cytometry
description	Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, are 30–800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.
format	article
author	Aizea Morales-Kastresana Bill Telford Thomas A. Musich Katherine McKinnon Cassandra Clayborne Zach Braig Ari Rosner Thorsten Demberg Dionysios C. Watson Tatiana S. Karpova Gordon J. Freeman Rosemarie H. DeKruyff George N. Pavlakis Masaki Terabe Marjorie Robert-Guroff Jay A. Berzofsky Jennifer C. Jones
author_facet	Aizea Morales-Kastresana Bill Telford Thomas A. Musich Katherine McKinnon Cassandra Clayborne Zach Braig Ari Rosner Thorsten Demberg Dionysios C. Watson Tatiana S. Karpova Gordon J. Freeman Rosemarie H. DeKruyff George N. Pavlakis Masaki Terabe Marjorie Robert-Guroff Jay A. Berzofsky Jennifer C. Jones
author_sort	Aizea Morales-Kastresana
title	Labeling Extracellular Vesicles for Nanoscale Flow Cytometry
title_short	Labeling Extracellular Vesicles for Nanoscale Flow Cytometry
title_full	Labeling Extracellular Vesicles for Nanoscale Flow Cytometry
title_fullStr	Labeling Extracellular Vesicles for Nanoscale Flow Cytometry
title_full_unstemmed	Labeling Extracellular Vesicles for Nanoscale Flow Cytometry
title_sort	labeling extracellular vesicles for nanoscale flow cytometry
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/fc12a330525a480593a5dd57e9326f33
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Labeling Extracellular Vesicles for Nanoscale Flow Cytometry

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