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, Karolinska Institutet researchers 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.
Multiplex bead-based flow cytometry assay principle
for detection of extracellular vesicle (EV) surface signatures
(A) Overview of assay workflow. 39 multiplexed populations of dye-labeled antibody-coated capture beads are incubated with EV-containing samples. In this case, captured EVs are counterstained with APC-labeled detection antibodies by using a mixture of anti-CD9, anti-CD63, and anti-CD81 (pan tetraspanin) antibodies. (B) Results after analyzing HEK293T conditioned medium (CM) compared to the respective medium control, showing all 39 bead populations identified by their fluorescence in the FITC Vs. PE channel (see Table S1 in Supplementary Material) with adjunct dot plots showing respective APC-stained bead populations. Back-gating from the four bead populations with the brightest staining (CD9, CD29, CD63, and CD81) is shown as an example to underline the assay principle (bottom right). (C) Representative quantification of the median APC fluorescence values for all bead populations after background correction (medium control values subtracted from measured HEK293T CM values).