Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

Abstract

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 order to 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, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

Figure 1. Schematic of the ASL pH measurement method.

After washing the cultures and performing a background reading, primary human airway epithelial cells (hAECs) ASL are loaded with dextran coupled pH-sensitive and pH-insensitive dye mixture overnight at 37 °C, 5% CO₂. The following day, the plate is transferred to a temperature and CO₂-controlled plate reader and fluorescence from both dyes is recorded over time. After the experiment, an in situ calibration is performed and data analyzed and presented as ASL pH over time.

Figure 2. Optimization of the pH calibration in vitro.

Different volumes of solutions of known pH and containing different dye concentrations were loaded onto a 96 well plate and fluorescence was recorded over 1 h. Effect of volume (A) and dye concentration (B) on fluorescence ratios. Ratios were plotted against pH for time-point 24 min. (C) The slope of change in fluorescence was calculated for each solution and plotted as a function of time (in min).

Figure 3. Optimization of the analysis of the pH calibration on primary hAECs in situ.

(A) Representative traces of ASL pH obtained from a single standard curve averaging data from non-CF and CF cultures. (B) Representative traces of ASL pH obtained from independent standard curve performed on non-CF or CF cultures. Each data set was calculated from its own calibration curve. (C) Evaluation of the differences in ASL pH between non-CF and CF cultures as a function of how the calibration was performed. Data represent the mean ± SEM from n=3 experiments, 2-way ANOVA, Sidak's multiple comparisons test).

Figure 4. Dynamic ASL pH measurement in response to CFTR activation by forskolin.

(A) Representative traces of the effect of forskolin (Fsk, 10 µM) on the kinetics of ASL pH over time in non-CF and CF hAECs. Data represent the mean ± SEM from n=3 experiments. (B) Summary of the effect of Fsk on ASL pH in non-CF and CF cultures. Data represent the mean ± SD from n=69 non-CF cultures and 35 CF cultures (2-way ANOVA, Sidak's multiple comparisons test). (C) Representative traces of the effect of CFTR_inh172 (172, 20 µM) on the Fsk-induced increase in ASL pH in non-CF hAECs. Data represent the mean ± SEM from n=5 experiments. (D) Summary of the effect of 172 on Fsk-induced alkalinisation of the ASL in non-CF cultures. Data represent the mean ± SEM from n=5 experiments (2-way ANOVA, Sidak's multiple comparisons test).

Figure 5. Dynamic changes in ASL pH in response to HCO₃^- removal.

(A) Representative traces showing the effect of HCO₃^- removal on the kinetics of ASL pH over time in non-CF and CF hAECs. The initial rates of acidification were obtained via the slope of a straight line fitted to 7 time-points after HCO₃^- removal. Data represent the means ± SEM from n=6 and 7 experiments on non-CF and CF cultures respectively. (B) Summary of the initial rates of acidification following HCO₃^- removal. Data represent the means ± SEM from n=6 and 7 experiments on non-CF and CF cultures respectively (Mann-Whitney test).