Dynamics of dendritic cell-derived vesicles: high-resolution flow cytometric analysis of extracellular vesicle quantity and quality

Abstract

Nano-sized membrane vesicles are secreted by many cell types. These vesicles can serve as carriers of cellular information. DC-derived vesicles can be targeted to other immune cells and modify their function. Accurate analysis of quantitative and qualitative changes in EV production by DC upon different activation stimuli is needed to further reveal the immune regulatory properties of DC-derived EVs. However, methods for reliable quantification of individual EVs and for analysis of the heterogeneity of EV populations are limited. With our recently developed highresolution flow cytometry-based method, we can perform a high-throughput, multiparameter, and quantitative analysis of individual EVs. With the use of this novel technique, we show that despite previous assumptions, stimulation with bacterial LPS increases EV release by DC. Furthermore, we demonstrate heterogeneity in DC-derived EVs regarding their buoyant density and MHC class II content. Finally, we show that cognate interaction between LPS-stimulated DC and CD4 ϩ T cells affects both the quantity and quality of LPS DC-derived EVs present in the culture supernatant. These data indicate that flow cytometry-based analysis of individual EVs is a valuable, novel tool to study the dynamics of EV secretion and composition, offering great opportunities to unveil the function of immune cell-derived EVs. J. Leukoc. Biol. 93: 395-402; 2013.

Extracellular vesicles shuffling intercellular messages: for good or for bad

“…A major limiting factor in the characterization of extracellular vesicles has been the absence of trustworthy methods for quantifying vesicle release. A reliable method to analyze small amounts of isolated exosomes is an optimized FACS procedure based on the labelling of isolated vesicles with fluorescent probes [14,15]. As a complement, the NanoSight LM10 based on light scattering automatically tracks and sizes nanoparticles on an individual basis although it is difficult to discriminate between small aggregates and vesicles [16].…”

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

“…More importantly, the dot plots in Figure 3D clearly illustrate a change in light scatter-based heterogeneity in EVs when measured at too high vesicle concentrations. Besides light scatter profile of EVs, we previously demonstrated high-resolution flow cytometric identification of different EV populations based on fluorescent antibody labeling (3,4,10). We here show that co-staining of CFSE-labeled mast cell-derived vesicles …”

“…The release of EVs and their content, i.e., proteins, lipids and RNAs, is tightly regulated and varies not only between different cell types but also depends on the physiological state of the producing cell (2)(3)(4). Consequently, EV release is very dynamic and the EV population is very heterogeneous.…”

“…3). This method has previously also facilitated the identification of activation status-dependent changes in EV subsets released by dendritic cells and T cells (3,4,10). However, to gain more insight in the composition and function of specific EV subsets and to identify EV-based biomarkers, accurate methods are needed for EV-subset isolation.…”

Therapeutic and diagnostic applications of extracellular vesicles

“…However, for diagnostic purposes such an approach will require screening of large amounts of vesicles as 'diseased' EVs are rare among the total isolated EV population. One promising approach relies on modifying flow cytometry equipment/protocols to detect single, nanosized EVs [244][245][246]. However, to date FACS is not able to detect the lower size range of EVs and requires antibodies (and hence also knowledge of a particular disease marker) to phenotype EVs.…”