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An accurate, precise method for general labeling of extracellular vesicles
Extracellular, membrane vesicles (microvesicles, exosomes) are secreted by cells and may serve as mediators of intercellular communication. Methods for detecting them by flow cytometry have included the use of agents that fluorescently stain vesicle membrane, or fluorescent antibodies that target sp...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589801/ https://www.ncbi.nlm.nih.gov/pubmed/26543819 http://dx.doi.org/10.1016/j.mex.2015.08.002 |
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author | Gray, Warren D. Mitchell, Adam J. Searles, Charles D. |
author_facet | Gray, Warren D. Mitchell, Adam J. Searles, Charles D. |
author_sort | Gray, Warren D. |
collection | PubMed |
description | Extracellular, membrane vesicles (microvesicles, exosomes) are secreted by cells and may serve as mediators of intercellular communication. Methods for detecting them by flow cytometry have included the use of agents that fluorescently stain vesicle membrane, or fluorescent antibodies that target specific cell-of-origin antigens. However, these methods may falsely detect cell debris or require prior cell-of-origin knowledge. Here, we demonstrate the suitability of calcein AM for detection of intact extracellular vesicles (EVs) by flow cytometry. • Calcein AM is non-fluorescent until it passively enters EVs, after which it is activated and becomes fluorescent and EV-impermeant. • Permeabilized/lysed EVs label positive with antibodies and lipophilic membrane stain, whereas no labeling was observed with calcein. In contrast to methods that use antibodies or membrane stains, calcein AM allows for the differentiation between intact EVs and debris. • Calcein AM can be used for detection of intact EVs from numerous cell types. |
format | Online Article Text |
id | pubmed-4589801 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-45898012015-11-05 An accurate, precise method for general labeling of extracellular vesicles Gray, Warren D. Mitchell, Adam J. Searles, Charles D. MethodsX Biochemistry, Genetics and Molecular Biology Extracellular, membrane vesicles (microvesicles, exosomes) are secreted by cells and may serve as mediators of intercellular communication. Methods for detecting them by flow cytometry have included the use of agents that fluorescently stain vesicle membrane, or fluorescent antibodies that target specific cell-of-origin antigens. However, these methods may falsely detect cell debris or require prior cell-of-origin knowledge. Here, we demonstrate the suitability of calcein AM for detection of intact extracellular vesicles (EVs) by flow cytometry. • Calcein AM is non-fluorescent until it passively enters EVs, after which it is activated and becomes fluorescent and EV-impermeant. • Permeabilized/lysed EVs label positive with antibodies and lipophilic membrane stain, whereas no labeling was observed with calcein. In contrast to methods that use antibodies or membrane stains, calcein AM allows for the differentiation between intact EVs and debris. • Calcein AM can be used for detection of intact EVs from numerous cell types. Elsevier 2015-09-10 /pmc/articles/PMC4589801/ /pubmed/26543819 http://dx.doi.org/10.1016/j.mex.2015.08.002 Text en http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Biochemistry, Genetics and Molecular Biology Gray, Warren D. Mitchell, Adam J. Searles, Charles D. An accurate, precise method for general labeling of extracellular vesicles |
title | An accurate, precise method for general labeling of extracellular vesicles |
title_full | An accurate, precise method for general labeling of extracellular vesicles |
title_fullStr | An accurate, precise method for general labeling of extracellular vesicles |
title_full_unstemmed | An accurate, precise method for general labeling of extracellular vesicles |
title_short | An accurate, precise method for general labeling of extracellular vesicles |
title_sort | accurate, precise method for general labeling of extracellular vesicles |
topic | Biochemistry, Genetics and Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589801/ https://www.ncbi.nlm.nih.gov/pubmed/26543819 http://dx.doi.org/10.1016/j.mex.2015.08.002 |
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