Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout

Abstract

Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells.However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs -in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification.

Quantitative analysis of protein-protein interactions and post-translational modifications in rare immune populations

“…Moreover, analysis of the fluorescent signal on tissue sections may be very inaccurate and problematic, as a result of very high variation among individual slides, often leading to over- or under-representation of a specific cell population in a given tissue section. As demonstrated by previous 28 – 30 and our studies, both PLA-FACS and PLIC are suitable for multiparametric analysis. However, unlike PLA-FACS, PLIC is much more accurate as it allows a very efficient filtering of auto-fluorescence and/or non-specific signals (caused by non-specific binding of the conjugated antibodies), by utilizing advanced features of the IFC imaging analysis including signal shape and localization.…”

“…2e , Supplementary Fig. 4 ), which would have been otherwise detected by conventional PLA protocol or PLA coupled to conventional flow cytometry 28 – 30 . This was further highlighted by similar PLIC analysis of Aire-deficient mTEC hi (Fig.…”

“…Furthermore, PLIC overcomes many key limitations of the currently available PLA-based methods. Although PLA has been used in combination with either confocal microscopy or conventional flow cytometry (PLA-FACS) 28 – 30 , the combination of PLA with IFC in PLIC enables a far more accurate, broader, and more sensitive detection of PPI or PTM than these previously described protocols. Specifically, as already discussed before, the key limitation of PLA followed by confocal microscopy is that it does not allow quantitative multi-parameter analysis of rare cells that are defined by multiple markers.…”

Multiplexed Profiling of Single Extracellular Vesicles

“…Recent approaches have included optical trapping, Raman spectroscopy, and flow cytometry. 18,19 However, multiplexed protein analysis in individual vesicles has been much more difficult.…”

The functional role of surface molecules on extracellular vesicles in cancer, autoimmune diseases, and coagulopathy

“…Flow cytometry, for example, is one of the most widely used method to detect EV surface proteins, but it is limited by the small sizes of EVs (exosomes particularly, as they are the smallest among EVs). 97 Newer methods involving the usage of microchips for EV array are being developed for multiplexed analysis of EV surface protein markers, which can expose the EVs to a panel of different protein markers, though currently only tetraspanins are relatively well tested. 69 A thorough evaluation of the use of microchips is needed in order for them to be widely used for detection of EV surface biomarkers with high sensitivity.…”