Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

Abstract

Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.

Microvesicles and exosomes: new players in metabolic and cardiovascular disease

“…Techniques are being developed which may even allow the detection of individual exosomes using dedicated flow cytometers with special labelling methods (Pospichalova et al 2015). An alternative and more widespread approach is to bind exosomes to carrier latex beads, which are easily detectable by flow cytometry (Thery et al 2006) (Fig.…”

Measurement of microvesicle levels in human blood using flow cytometry

“…In summary, measurement of microvesicles in blood requires either a flow cytometer designed to measure microvesicles if light scattering or impedance is used (41,47,64) or fluorescence triggering if a conventional flow cytometer is used. Background noise and bead size resolution will need to be determined for the method used to trigger a microvesicle counts, followed by approximate calibration of the flow cytometer for microvesicle size.…”

Characterization of extracellular vesicles by IR spectroscopy: Fast and simple classification based on amide and C H stretching vibrations

“…Beside current techniques like electron microscopy (cryo-EM or freeze-fracture combined EM) [11,12], flow cytometry [13,14], and dynamic light scattering (DLS) [15], the introduction of novel techniques like nanoparticle tracking analysis (NTA) [16], resistive pulse sensing (RPS), small-angle X-ray scattering (SAXS) or size exclusion chromatography coupled with dynamic light scattering (SEC-DLS) are also ongoing [17,18]. Another key issue in EV research is the total protein determination and characterization by bioanalytical assays [19][20][21]. Quite recently, the protein-to-lipid ratio measured by combined BCA protein assay and sulfophosphovanillin (SPV) lipid assay as an additional parameter for routine quality control of EV preparation was proposed [22].…”