- Article
- Open access
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- Georgina Hopkins1 na1,
- William Browne1 na1,
- Davis Tucis1 na1,
- Stella Cochrane2,
- Victoria James3,
- David Onion1 &
- …
- Lucy C. Fairclough1
Scientific Reports , Article number: (2026) Cite this article
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Abstract
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.
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All data required to evaluate the conclusions in the paper are present in the manuscript or its supplementary material. Further information is available from the corresponding authors on reasonable request.
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Acknowledgements
We thank Beckman Coulter and the University of Birmingham, UK, for providing access to the CytoFLEX nano flow cytometer, housed at the University of Birmingham’s Medical School.
Funding
Author GH was funded by Unilever (Grant Ref MA-2023–00556 N). Author WB was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant Ref BB/W510506/1) and Unilever (Grant Ref MA-2020–02076 N). The ImageStream X MKII was funded by the Wellcome to LF and DO (Grant Ref 212908/Z/18/Z).
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Competing interests
Author SC was employed by Unilever’s Safety Environmental and Regulatory Science Centre.
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Hopkins, G., Browne, W., Tucis, D. et al. Bronchial epithelial cell-derived extracellular vesicle analysis using conventional, imaging, and nanoscale flow cytometry technologies. Sci Rep (2026). https://doi.org/10.1038/s41598-026-41848-x
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DOI: https://doi.org/10.1038/s41598-026-41848-x
