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Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV...

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Autores principales: Ender, Fanny, Zamzow, Piet, von Bubnoff, Nikolas, Gieseler, Frank
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981603/
https://www.ncbi.nlm.nih.gov/pubmed/31906247
http://dx.doi.org/10.3390/ijms21010291
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author Ender, Fanny
Zamzow, Piet
von Bubnoff, Nikolas
Gieseler, Frank
author_facet Ender, Fanny
Zamzow, Piet
von Bubnoff, Nikolas
Gieseler, Frank
author_sort Ender, Fanny
collection PubMed
description The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.
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spelling pubmed-69816032020-02-03 Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters! Ender, Fanny Zamzow, Piet von Bubnoff, Nikolas Gieseler, Frank Int J Mol Sci Article The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies. MDPI 2019-12-31 /pmc/articles/PMC6981603/ /pubmed/31906247 http://dx.doi.org/10.3390/ijms21010291 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ender, Fanny
Zamzow, Piet
von Bubnoff, Nikolas
Gieseler, Frank
Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!
title Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!
title_full Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!
title_fullStr Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!
title_full_unstemmed Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!
title_short Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!
title_sort detection and quantification of extracellular vesicles via facs: membrane labeling matters!
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981603/
https://www.ncbi.nlm.nih.gov/pubmed/31906247
http://dx.doi.org/10.3390/ijms21010291
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