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A cell-based assay for CD63-containing extracellular vesicles

Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for moni...

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Detalles Bibliográficos
Autores principales: Cashikar, Anil G., Hanson, Phyllis I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6655660/
https://www.ncbi.nlm.nih.gov/pubmed/31339911
http://dx.doi.org/10.1371/journal.pone.0220007
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author Cashikar, Anil G.
Hanson, Phyllis I.
author_facet Cashikar, Anil G.
Hanson, Phyllis I.
author_sort Cashikar, Anil G.
collection PubMed
description Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.
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spelling pubmed-66556602019-08-07 A cell-based assay for CD63-containing extracellular vesicles Cashikar, Anil G. Hanson, Phyllis I. PLoS One Research Article Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion. Public Library of Science 2019-07-24 /pmc/articles/PMC6655660/ /pubmed/31339911 http://dx.doi.org/10.1371/journal.pone.0220007 Text en © 2019 Cashikar, Hanson http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Cashikar, Anil G.
Hanson, Phyllis I.
A cell-based assay for CD63-containing extracellular vesicles
title A cell-based assay for CD63-containing extracellular vesicles
title_full A cell-based assay for CD63-containing extracellular vesicles
title_fullStr A cell-based assay for CD63-containing extracellular vesicles
title_full_unstemmed A cell-based assay for CD63-containing extracellular vesicles
title_short A cell-based assay for CD63-containing extracellular vesicles
title_sort cell-based assay for cd63-containing extracellular vesicles
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6655660/
https://www.ncbi.nlm.nih.gov/pubmed/31339911
http://dx.doi.org/10.1371/journal.pone.0220007
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