Abstract
Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.
Highlights
Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids
After incubation with EV-containing samples, bulk bead-captured EVs can subsequently be detected by counterstaining with APC-labeled detection antibodies against the tetraspanins CD9, CD63, and CD81, which are often referred to as common EV surface markers
The results presented in this study demonstrate that this assay facilitates EV surface marker detection in different types of samples in a very specific and reproducible way
Summary
Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids They can be roughly classified based on their size and subcellular origin as exosomes (70–150 nm in diameter) which are released when multivesicular bodies fuse with the plasma membrane [1], or microvesicles (100 nm to 1 μm in diameter) which are formed by the outward budding of the plasma membrane [2, 3]. In addition to these different EV subtypes, nowadays it is accepted in the field that there is likely to be a much higher degree of EV heterogeneity at multiple levels within each subentity [reviewed in Ref. The identification of specific vesicular surface markers will be of great relevance to further understand the molecular content and related functions of subsets of EVs, to identifying potential EV subsets with a defined therapeutic activity, and to uncovering and defining specific disease-related biomarkers
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