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.
