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

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

Scientific Reports , Article number:  (2026) Cite this article

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

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.

Similar content being viewed by others

Data availability

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.

References

  1. Welsh, J. A. et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J. Extracell. Vesicles. 13 (2), e12404 (2024).

    Google Scholar 

  2. Spitzberg, J. D. et al. Multiplexed analysis of EV reveals specific biomarker composition with diagnostic impact. Nat. Commun. 14 (1), 1239 (2023).

    Google Scholar 

  3. Browne, W. et al. The Role of Epithelial-Derived Extracellular Vesicles in Allergic Sensitisation: A Systematic Review. Int. J. Mol. Sci. 26(12), 5791 (2025).

  4. Tucis, D. et al. The Role of Extracellular Vesicles in Allergic Sensitization: A Systematic Review. Int. J. Mol. Sci. 25 (8), 4492 (2024).

    Google Scholar 

  5. Sarkar, S. et al. Extracellular vesicles secreted by primary human bronchial epithelial cells reduce Pseudomonas aeruginosa burden and inflammation in cystic fibrosis mouse lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 326 (2), L164–l174 (2024).

    Google Scholar 

  6. Xu, H. et al. Exosomal microRNA-21 derived from bronchial epithelial cells is involved in aberrant epithelium-fibroblast cross-talk in COPD induced by cigarette smoking. Theranostics 8 (19), 5419–5433 (2018).

    Google Scholar 

  7. Kadota, T. et al. Human bronchial epithelial cell-derived extracellular vesicle therapy for pulmonary fibrosis via inhibition of TGF-β-WNT crosstalk. J. Extracell. Vesicles. 10 (10), e12124 (2021).

    Google Scholar 

  8. Fraikin, J. L. et al. A high-throughput label-free nanoparticle analyser. Nat. Nanotechnol. 6 (5), 308–313 (2011).

    Google Scholar 

  9. van der Pol, E. et al. Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles. J. Thromb. Haemost. 14 (1), 48–56 (2016).

    Google Scholar 

  10. Gul, B. et al. Characterization of extracellular vesicles by flow cytometry: Challenges and promises. Micron 161, 103341 (2022).

    Google Scholar 

  11. Zhu, S. et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS Nano. 8 (10), 10998–11006 (2014).

    Google Scholar 

  12. Kim, J. et al. Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer. J. Extracell. Vesicles. 13 (8), e12498 (2024).

    Google Scholar 

  13. Chuo, S. T. Y., Chien, J. C. Y. & Lai, C. P. K. Imaging extracellular vesicles: current and emerging methods. J. Biomed. Sci. 25 (1), 91 (2018).

    Google Scholar 

  14. Gray, W. D., Mitchell, A. J. & Searles, C. D. An accurate, precise method for general labeling of extracellular vesicles. MethodsX 2, 360–367 (2015).

    Google Scholar 

  15. Brealey, J. et al. Shining a light on fluorescent EV dyes: Evaluating efficacy, specificity and suitability by nano-flow cytometry. J. Extracell. Biol. 3 (10), e70006 (2024).

    Google Scholar 

  16. Chen, C. et al. Quantitative assessment of lipophilic membrane dye-based labelling of extracellular vesicles by nano-flow cytometry. J. Extracell. Vesicles. 12 (8), e12351 (2023).

    Google Scholar 

  17. Haines, L. A. et al. Non-Specific Particle Formation During Extracellular Vesicle Labelling With the Lipophilic Membrane Dye PKH26. J. Extracell. Vesicles. 14 (5), e70079 (2025).

    Google Scholar 

  18. Welsh, J. A. et al. MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. J. Extracell. Vesicles. 9 (1), 1713526 (2020).

    Google Scholar 

  19. Woud, W. W. et al. An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma. Commun. Biology. 5 (1), 633 (2022).

    Google Scholar 

  20. Barranco, I. et al. Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma. Cell. Communication Signal. 22 (1), 63 (2024).

    Google Scholar 

  21. Görgens, A. et al. Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material. J. Extracell. Vesicles. 8 (1), 1587567 (2019).

    Google Scholar 

  22. Mizenko, R. R. et al. Tetraspanins are unevenly distributed across single extracellular vesicles and bias sensitivity to multiplexed cancer biomarkers. J. Nanobiotechnol. 19 (1), 250 (2021).

    Google Scholar 

  23. Schindler, V. E. M. et al. Side-Directed Release of Differential Extracellular Vesicle-associated microRNA Profiles from Bronchial Epithelial Cells of Healthy and Asthmatic Subjects. Biomedicines 10 (3), 622 (2022).

    Google Scholar 

  24. Tertel, T. et al. Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell-extracellular vesicle preparations. Cytotherapy 24 (6), 619–628 (2022).

    Google Scholar 

  25. Wiklander, O. P. B. et al. Antibody-displaying extracellular vesicles for targeted cancer therapy. Nat. Biomedical Eng. 8(11), 1453–1468 (2024).

  26. Pasalic, L. et al. Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis. Nanomed. Nanotechnol. Biol. Med. 12 (4), 977–986 (2016).

    Google Scholar 

  27. Welsh, J. A. et al. FCM(PASS) Software Aids Extracellular Vesicle Light Scatter Standardization. Cytometry A. 97 (6), 569–581 (2020).

    Google Scholar 

  28. Welsh, J. A. & Jones, J. C. Small Particle Fluorescence and Light Scatter Calibration Using FCMPASS Software. Curr. Protocols Cytometry. 94 (1), e79 (2020).

    Google Scholar 

  29. Yung, C. et al. Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid. J. Extracell. Vesicles. 13 (4), e12422 (2024).

    Google Scholar 

  30. Welsh, J. A. et al. Extracellular Vesicle Flow Cytometry Analysis and Standardization. Front. Cell. Dev. Biology, 5. (2017).

  31. Sokolova, V. et al. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf. B Biointerfaces. 87 (1), 146–150 (2011).

    Google Scholar 

  32. Lucchetti, D. et al. Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality? Int. J. Mol. Sci. 21 https://doi.org/10.3390/ijms21176257 (2020).

  33. McVey, M. J., Spring, C. M. & Kuebler, W. M. Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter. J. Extracell. Vesicles. 7 (1), 1454776 (2018).

    Google Scholar 

  34. Negahdaripour, M. et al. Small extracellular vesicles (sEVs): discovery, functions, applications, detection methods and various engineered forms. Expert Opin. Biol. Ther. 21 (3), 371–394 (2021).

    Google Scholar 

  35. Momen-Heravi, F. et al. Alternative Methods for Characterization of Extracellular Vesicles Volume 3–2012 (Frontiers in Physiology, 2012).

  36. Calderón-Peláez, M. A., Castellanos, J. E. & Velandia-Romero, M. L. A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system. PLoS One. 20 (1), e0317689 (2025).

    Google Scholar 

  37. de Rond, L. et al. Comparison of Generic Fluorescent Markers for Detection of Extracellular Vesicles by Flow Cytometry. Clin. Chem. 64 (4), 680–689 (2018).

    Google Scholar 

  38. Liao, Z. et al. Acetylcholinesterase is not a generic marker of extracellular vesicles. J. Extracell. Vesicles. 8 (1), 1628592 (2019).

    Google Scholar 

  39. Kugeratski, F. G. et al. Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker. Nat. Cell Biol. 23 (6), 631–641 (2021).

    Google Scholar 

  40. Nazarenko, I. et al. Cell Surface Tetraspanin Tspan8 Contributes to Molecular Pathways of Exosome-Induced Endothelial Cell Activation. Cancer Res. 70 (4), 1668–1678 (2010).

    Google Scholar 

  41. Okada-Tsuchioka, M. et al. Tetraspanin heterogeneity of small extracellular vesicles in human biofluids and brain tissue. Biochem. Biophys. Res. Commun. 627, 146–151 (2022).

    Google Scholar 

  42. Hemler, M. E. Tetraspanin Proteins Mediate Cellular Penetration, Invasion, and Fusion Events and Define a Novel Type of Membrane Microdomain. Annu. Rev. Cell Dev. Biol. 19 (1), 397–422 (2003).

    Google Scholar 

Download references

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).

Author information

Author notes

  1. These authors contributed equally to this work: Georgina Hopkins, William Browne and Davis Tucis.

Authors and Affiliations

  1. School of Life Sciences, The University of Nottingham, Nottingham, NG7 2UH, UK

    Georgina Hopkins, William Browne, Davis Tucis, David Onion & Lucy C. Fairclough

  2. SERS, Unilever, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK

    Stella Cochrane

  3. School of Veterinary Medicine and Science, The University of Nottingham, Nottingham, NG7 2UH, UK

    Victoria James

Authors

  1. Georgina Hopkins
  2. William Browne
  3. Davis Tucis
  4. Stella Cochrane
  5. Victoria James
  6. David Onion
  7. Lucy C. Fairclough

Contributions

GH, WB, and DT performed all experiments and conducted data analysis. GH wrote the manuscript and prepared figures, with sections contributed by WB and DT. SC, VJ, DO, and LF reviewed and edited the manuscript. LF and DO designed the study, acquired the funding, and LF managed the project.

Corresponding author

Correspondence to Lucy C. Fairclough.

Ethics declarations

Competing interests

Author SC was employed by Unilever’s Safety Environmental and Regulatory Science Centre.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41598-026-41848-x

Keywords