Abstract
The respiratory epithelium is the initial point of host contact for inhaled particles, leading to orchestrated, but highly heterogeneous, responses. Human airway epithelial cells (AECs) play a crucial role in host defense by promoting uptake and killing of inhaled microorganisms and concomitant cytokine production in order to recruit professional phagocytes to the site of infection. However, inhaled pathogens can also reside and replicate intracellularly to evade host immune defenses or circulating antimicrobial drugs, ultimately causing apoptosis or cell death of the infected AECs. Imaging flow cytometry (IFC) combines flow cytometry, fluorescent microscopy, and advanced data-processing algorithms to dissect the heterogeneity of the interaction of AECs and inhaled microorganisms and its outcomes at the single-cell level. Here, we describe a novel single-cell approach based on differential fluorescent staining and state-of-the-art IFC to identify, quantify, and analyze individual host–pathogen complexes from cultured AECs infected with spores of the major human fungal pathogen Aspergillus fumigatus.
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Acknowledgments
This work was supported by grants to Prof. Elaine M. Bignell (University of Manchester) from the Medical Research Council (G0501164, MR/L000822/1 and MR/M02010X/1), the Biotechnology and Biological Sciences Research Council (BB/G009619/1) and the Wellcome Trust (WT093596MA) and the Chelsea and Westminster Healthcare National Health Service Trust Charity, to M.B. from Imperial College London (Division of Investigative Sciences PhD Studentships) and Fungal Infection Trust and to E.M.B. and M.B. from a University of Manchester Medical Research Council Discovery Award (MC_PC_15072).
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Bertuzzi, M., Howell, G.J. (2021). Single-Cell Analysis of Fungal Uptake in Cultured Airway Epithelial Cells Using Differential Fluorescent Staining and Imaging Flow Cytometry. In: Bignell, E. (eds) Host-Fungal Interactions. Methods in Molecular Biology, vol 2260. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1182-1_6
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DOI: https://doi.org/10.1007/978-1-0716-1182-1_6
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