Bronchial epithelial cell-derived extracellular vesicle analysis using conventional, imaging, and nanoscale flow cytometry technologies.

Bronchial epithelial cell-derived extracellular vesicles (EVs) are central to airway immune responses to inhaled particulate stimuli, as well as regulating respiratory diseases. Through a complex bioactive cargo, EVs can influence inflammatory signalling in the epithelium. Typically, a variety of technologies are used to analyse EVs derived from biofluids, such as nanoparticle tracking analysis, western blotting, and transmission electron microscopy. But recent advances in flow cytometers (FCs) potentially provide a single technology that can rapidly enumerate, size and phenotype epithelial cell-derived EVs, without the absolute need for purification. With multiple FCs available, this study aimed to describe methods and discuss considerations for analysing epithelial cell-derived EV on different FCs. Thus, supernatants containing EVs from primary human bronchial epithelial cells were stained with calcein-AM, in combination with anti-fluorescently conjugated tetraspanin antibodies, before analysing on a CytoFLEX S, ImageStream X MKII, and CytoFLEX nano. NIST traceable polystyrene particle size standards or synthetic EV size standards were used for EV size calibration, and antibody capture microspheres were used to measure the limit of detection for tetraspanin antibodies. We demonstrated that epithelial cell-derived EVs can be sized, enumerated, and phenotyped using all tested FC technologies, with varying sizing sensitivities and considerations for each FC. These findings provide a guidance for selecting suitable FC technologies for EV characterisation and highlight their potential to dissect epithelial EV heterogeneity in the context of airway immune responses and inflammation.