The more the better – determining the optimal range when performing single-vesicle phenotyping
DOI: https://doi.org/10.47184/tev.2021.01.03 The characterization of extracellular vesicles (EVs) has evolved rapidly in recent years due to advances in straightforward technologies. Based on these more sensitive methods, it is now possible to describe EV populations in their entirety more precisely. However, these applications require an equivalently delicate experiment design and optimization steps to draw valid conclusions in the end. One of these methods is represented by the highly sensitive nanoflow cytometry (nFCM), by which particles can be analyzed not only on their size (< 40 nm) and concentration but also concerning surface markers. In this work, we addressed some of the potential caveats of this method, especially when characterizing particles with fluorescently labelled antibodies. In particular, we show, when using low particle concentrations, which are inevitably encountered when working with EVs, the characterization of surface markers is prone to significantly varying. We hypothesized that these technical limitations could respond to the stickiness of EVs and should be properly counteracted. As a reference, we strongly recommend performing particle number-based comparisons with at least 109 particles as staining input in nFCM analyses. Moreover, we provided representative particle-number based immunoblotting results, underlying the significance of this parameter as a normalizer in future EV research.
Extracellular vesicles, size-exclusion chromatography, single-vesicle fluorescence analysis, standardization
Introduction
Extracellular vesicles (EVs) are omnipresent lipid particles released by all cells and are considered to play an important role in cell-to-cell communication under both physiological as well as pathological conditions. Among others, microvesicles that directly bud from the plasma membrane and exosomes, which originate from the endosomal compartment, represent the two major types of EVs that have attracted the attention of the scientific community in recent years. Although, both EV subtypes can be distinguished by their source of origin in theory, they also share common biophysical and biochemical peculiarities such as size and markers [for review see 1].
To improve the characterization of the various EV subtypes, several technologies have been developed in recent years [2, 3], which allow for the phenotyping of single particles by individually detecting specific surface molecules. On the one hand, this methodology increases reproducibility as on the other hand, it allows a more detailed description of the distinct EVs compared to the early years of EV characterization. Flow cytometry (FC) approaches have been predominantly used to characterize EVs in detail. However, some drawbacks of standard FC for EV analyses include but are not limited to inaccurate sizing estimation, the necessity of suitable instrumentation as well a certain level of expertise [4].
The progression of standard FC to nanoflow range (also referred to as, nano flow cytometry, nFCM) has significantly improved the characterization of EVs in terms of their size by single-particle measurements by circumventing the detection of so-called swarm events, which are often detected in conventional flow cytometry when analysing EVs [3, 5]. EV research and its representatives by the International Society for Extracellular Vesicles (ISEV) have pointed out several times the importance of reproducibility and standardization, especially when novel approaches raised and need to be implemented in different settings [4]. Thus, in this work, we aim to contribute to the description of some important technical issues observed when performing fluorescence analyses by nFCM to reduce any potential bias in future research. In particular, we have demonstrated that the total particle number used as input for the analysis can influence the percentage of positive events detected by nFCM (output) under certain staining conditions, independently of the surface marker analysed.
Finally, in this technical note we have also included a serial dilution of particles for the detection of typical intravesicular markers such as Alix, flotillin-1 (FLOT-1), and TSG101, by SDS-PAGE, in which a limiting particle number showed an equivalent detection level compared to nFCM analyses. Low particle yields are a common limitation in EV research, especially when working with primary material, becoming an important target for confounding and variable results. When EV release is impaired under certain treatments compared to controls, comparative fluorescence nFCM analysis could introduce a technical bias if comparisons are based on volume : volume proceedings instead of total particle number even for cell culture models. In sum, our results have shown that the more particles used as input material, the more reliable results we obtain, indicating an unexpected variable that needs to be critically considered in single-EV fluorescence analyses and to be implemented in the field for the upcoming years.
Results and discussion
Purification and initial characterization of ES-2 EVs
For validating the performance of nFCM, we isolated EVs from the ovarian clear-cell carcinoma cell line ES-2. We specifically selected this cell line because, according to our experience, we achieve relatively high quantities of EVs on which, in addition, the classic EV markers CD9, CD63 and CD81 can be detected quite efficiently using (nano) FC. EVs were isolated using a combination of ultra/centrifugation and size-exclusion chromatography (SEC) (Figure 1A).
