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 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.

Automated summary

The quantification of disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened new opportunities for liquid biopsy, but the sensitivity is still not optimal for routine clinical application. This study developed a new approach to non-specific ENV staining and SEC fractionation to increase the sensitivity of bead-assisted flow cytometry. The process of CM-Dil-stained plasma fractionation was characterized in detail, and the fractions with optimal characteristics were identified, while also exploring the sensitivity of on-bead flow cytometry for specific populations of plasma ENVs and demonstrating the advantages and limitations of the technique.