Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Nikiforova N

PROVIDER: S-EPMC8303825 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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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.

Nikiforova Nadezhda N Chumachenko Maria M Nazarova Inga I Zabegina Lidia L Slyusarenko Maria M Sidina Elena E Malek Anastasia A

Membranes 20210713 7

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 ...[more]

PMID: 34357175

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Project description:BackgroundHuman umbilical cord mesenchymal stem cell (hUC-MSC) therapy is considered as a promising approach in the treatment of intrauterine adhesions (IUAs). Considerable researches have already detected hUC-MSCs by diverse methods. This paper aims at exploring the quantitative distribution of CM-Dil-labeled hUC-MSCs in different regions of the uterus tissue of the dual injury-induced IUAs in rats and the underlying mechanism of restoration of fertility after implantation of hUC-MSCs in the IUA model.MethodsIn this study, we investigated the quantification of the CM-Dil-labeled hUC-MSCs migrated to the dual injured uterus in Sprague Dawley rats. Additionally, we investigated the differentiation of CM-Dil-labeled hUC-MSCs. The differentiation potential of epithelial cells, vascular endothelial cells, and estrogen receptor (ER) cells were assessed by an immunofluorescence method using CK7, CD31, and ERα. The therapeutic impact of hUC-MSCs in the IUA model was assessed by hematoxylin and eosin, Masson, immunohistochemistry staining, and reproductive function test. Finally, the expression of TGF-β1/Smad3 pathway in uterine tissues was determined by qRT-PCR and Western blotting.ResultsThe CM-Dil-labeled cells in the stroma region were significantly higher than those in the superficial myometrium (SM) (71.67 ± 7.98 vs. 60.92 ± 3.96, p = 0.005), in the seroma (71.67 ± 7.98 vs. 23.67 ± 8.08, p = 0.000) and in the epithelium (71.67 ± 7.98 vs. 4.17 ± 1.19, p = 0.000). From the 2nd week of treatment, hUC-MSCs began to differentiate into epithelial cells, vascular endothelial cells, and ER cells. The therapeutic group treated with hUC-MSCs exhibited a significant decrease in fibrosis (TGF-β1/Smad3) as well as a significant increase in vascularization (CD31) compared with the untreated rats.ConclusionOur findings suggested that the distribution of the migrated hUC-MSCs in different regions of the uterine tissue was unequal. Most cells were in the stroma and less were in the epithelium of endometrium and gland. Injected hUC-MSCs had a capacity to differentiate into epithelial cells, vascular endothelial cells, and ER cells; increase blood supply; inhibit fibration; and then restore the fertility of the IUA model.

Dataset Information

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

Publications

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

Similar Datasets

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