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Lookup NU author(s): Dr Vinciane Saint-CriqORCiD, Dr Iram Haq, Dr Aaron Ions GardnerORCiD, Professor Christopher WardORCiD, Dr Malcolm Brodlie, Dr Michael Gray
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in orderto ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored.To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, aswell as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.
Author(s): Saint-Criq V, Haq IJ, Gardner AI, Garnett JP, Ward C, Brodlie M, Gray MA
Publication type: Article
Publication status: Published
Journal: Journal of Visualized Experiements
Year: 2019
Issue: 148
Online publication date: 13/06/2019
Acceptance date: 25/03/2019
Date deposited: 25/06/2019
ISSN (electronic): 1940-087X
Publisher: Jove
URL: https://doi.org/10.3791/59815
DOI: 10.3791/59815
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