Labeling Extracellular Vesicles for Nanoscale Flow Cytometry

TLDR

Extracellular vesicles (30–800 nm) are released by most cell types and function as intercellular communication packages; their involvement in cancer and inflammation has positioned them as promising biomarkers and therapeutic agents, yet high‑resolution cytometry requires bright, artifact‑free fluorescent labels for single‑vesicle detection. The study aims to identify a robust strategy for fluorescently labeling individual extracellular vesicles to enable their visualization and detection by high‑resolution flow cytometry. Using nanoFACS, the authors compared lipid, protein, and RNA staining approaches, developed a robust amine‑reactive dye (5‑(and‑6)-Carboxyfluorescein Diacetate Succinimidyl Ester) protocol with size‑exclusion chromatography cleanup, and assessed labeling sensitivity and specificity via combined light‑scattering and fluorescence measurements. The authors demonstrated that nanoFACS‑based labeling with the amine‑reactive dye and size‑exclusion cleanup yields highly sensitive and specific detection of individual EVs, enabling efficient characterization that facilitates future studies of EV biology.

Abstract

Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, are 30–800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.