Abstract
Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.
Highlights
Extracellular vesicles (EVs) are membrane-surrounded structures released in the intercellular environment and blood stream by a large variety of cells
In the era of precision medicine, considerable interest using flow cytometry exists to assess both quantitative and phenotypic EVs characteristics in patients suffering from a variety of diseases in the hope to reveal clinically relevant EVs subpopulations
We investigated in depth whether small/medium-sized EVs (50 to 250 nm size range) can be detected by a conventional flow cytometer (CytoFLEX S, Beckman Coulter) designed primarily for common applications, mostly cell immunophenotyping
Summary
Extracellular vesicles (EVs) are membrane-surrounded structures released in the intercellular environment and blood stream by a large variety of cells. EVs shuttle lipids, proteins, RNA, DNA, and other metabolites between cells and tissues They diverge into two main subgroups according to their biogenesis and release mechanism: microvesicles (150–1000 nm in diameter, MVs) shed from the plasma membrane, and exosomes, which are generally smaller in size (30–150 nm in diameter, EXOs) originating from the endosome as intraluminal vesicles enclosed within multivesicular bodies [1]. A number of techniques are potentially suited to assess individual EVs, including electron microscopy, resistive pulse sensing, nanoparticle tracking analysis, dynamic light scatter (DLS), and flow cytometry [3]. Only the latter technique is able to combine high-throughput and adequate speed allowing EVs evaluation in translational studies and in a routine clinical setting. It is in the clinical setting that EVs are currently most extensively investigated by flow cytometry in a variety of pathological processes
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