Development of a quantitative method to measure EV uptake

Abstract The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrim...

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Autores principales:	Víctor Toribio, Sara Morales, Soraya López-Martín, Beatriz Cardeñes, Carlos Cabañas, María Yáñez-Mó
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2019
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ce856eb937584cc783e3dd5e39eccf02
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spelling	oai:doaj.org-article:ce856eb937584cc783e3dd5e39eccf022021-12-02T15:09:46ZDevelopment of a quantitative method to measure EV uptake10.1038/s41598-019-47023-92045-2322https://doaj.org/article/ce856eb937584cc783e3dd5e39eccf022019-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-019-47023-9https://doaj.org/toc/2045-2322Abstract The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.Víctor ToribioSara MoralesSoraya López-MartínBeatriz CardeñesCarlos CabañasMaría Yáñez-MóNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 9, Iss 1, Pp 1-14 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Víctor Toribio Sara Morales Soraya López-Martín Beatriz Cardeñes Carlos Cabañas María Yáñez-Mó Development of a quantitative method to measure EV uptake
description	Abstract The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.
format	article
author	Víctor Toribio Sara Morales Soraya López-Martín Beatriz Cardeñes Carlos Cabañas María Yáñez-Mó
author_facet	Víctor Toribio Sara Morales Soraya López-Martín Beatriz Cardeñes Carlos Cabañas María Yáñez-Mó
author_sort	Víctor Toribio
title	Development of a quantitative method to measure EV uptake
title_short	Development of a quantitative method to measure EV uptake
title_full	Development of a quantitative method to measure EV uptake
title_fullStr	Development of a quantitative method to measure EV uptake
title_full_unstemmed	Development of a quantitative method to measure EV uptake
title_sort	development of a quantitative method to measure ev uptake
publisher	Nature Portfolio
publishDate	2019
url	https://doaj.org/article/ce856eb937584cc783e3dd5e39eccf02
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Development of a quantitative method to measure EV uptake

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