Measuring extracellular vesicles by conventional flow cytometry: Dream or reality?

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.