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CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry

The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the is...

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Autores principales: Nikiforova, Nadezhda, Chumachenko, Maria, Nazarova, Inga, Zabegina, Lidia, Slyusarenko, Maria, Sidina, Elena, Malek, Anastasia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303825/
https://www.ncbi.nlm.nih.gov/pubmed/34357175
http://dx.doi.org/10.3390/membranes11070526
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author Nikiforova, Nadezhda
Chumachenko, Maria
Nazarova, Inga
Zabegina, Lidia
Slyusarenko, Maria
Sidina, Elena
Malek, Anastasia
author_facet Nikiforova, Nadezhda
Chumachenko, Maria
Nazarova, Inga
Zabegina, Lidia
Slyusarenko, Maria
Sidina, Elena
Malek, Anastasia
author_sort Nikiforova, Nadezhda
collection PubMed
description The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the isolation and labeling approaches, and is not yet sufficient for routine clinical application. The presented study aimed to develop, characterize, and explore a new approach to non-specific ENV staining, followed by size-exclusive chromatography (SEC), which allows us to increase the sensitivity of bead-assisted flow cytometry. Plasma from healthy donors was purified from large components, stained with lipophilic CM-Dil dye, and fractionated by means of SEC. The obtained fractions were analyzed in terms of particle size and concentration using NTA, as well as vesicular markers and plasma protein content via dot-blotting. We characterized the process of CM-Dil-stained plasma fractionation in detail and indicated the fractions with optimal characteristics. Finally, we explored the sensitivity of on-bead flow cytometry for the analysis of specific populations of plasma ENVs and demonstrated the advantages and limitations of the proposed technique.
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spelling pubmed-83038252021-07-25 CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry Nikiforova, Nadezhda Chumachenko, Maria Nazarova, Inga Zabegina, Lidia Slyusarenko, Maria Sidina, Elena Malek, Anastasia Membranes (Basel) Article The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the isolation and labeling approaches, and is not yet sufficient for routine clinical application. The presented study aimed to develop, characterize, and explore a new approach to non-specific ENV staining, followed by size-exclusive chromatography (SEC), which allows us to increase the sensitivity of bead-assisted flow cytometry. Plasma from healthy donors was purified from large components, stained with lipophilic CM-Dil dye, and fractionated by means of SEC. The obtained fractions were analyzed in terms of particle size and concentration using NTA, as well as vesicular markers and plasma protein content via dot-blotting. We characterized the process of CM-Dil-stained plasma fractionation in detail and indicated the fractions with optimal characteristics. Finally, we explored the sensitivity of on-bead flow cytometry for the analysis of specific populations of plasma ENVs and demonstrated the advantages and limitations of the proposed technique. MDPI 2021-07-13 /pmc/articles/PMC8303825/ /pubmed/34357175 http://dx.doi.org/10.3390/membranes11070526 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Nikiforova, Nadezhda
Chumachenko, Maria
Nazarova, Inga
Zabegina, Lidia
Slyusarenko, Maria
Sidina, Elena
Malek, Anastasia
CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry
title CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry
title_full CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry
title_fullStr CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry
title_full_unstemmed CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry
title_short CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry
title_sort cm-dil staining and sec of plasma as an approach to increase sensitivity of extracellular nanovesicles quantification by bead-assisted flow cytometry
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303825/
https://www.ncbi.nlm.nih.gov/pubmed/34357175
http://dx.doi.org/10.3390/membranes11070526
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