Our protocol is the first that allows the dynamic measurement of airway surface liquid, or ASL pH, in real-time under both resting and after agonist or inhibitor treatment. This method helps to identify ion channels and transporters involved in extracellular pH regulation and could be applied to other cell systems in which this is important, such as cancer. Now compared to previously published methods, this new technique is relatively simple to perform.
It doesn't require specialized equipment. And can make ASL pH measurements simultaneously in multiple cultures under air liquid interface, or thin film conditions. Importantly, this new technique has the potential to assess the effects of new therapeutics, not only for cystic fibrosis, but also for other chronic airway diseases, such as asthma and COPD.
After at least 28 days of culture, wash the apical surface of a human airway epithelial cell culture with 150 microliters of bicarbonate containing Krebs buffer solution for 20 minutes on the cell culture incubator. At the end of the incubation, use a sterile glass pipet with a sterile P200 pipet tip linked to an aspiration pump, to carefully aspirate the wash without disrupting the epithelium. There should be as little liquid remaining on the apical surface as possible to restore the air liquid interface.
Then return the cells to the cell culture incubator for 30 minutes. To perform a background measurement, turn on the plate reader and the computer. After opening the dashboard, click Spark 10M and set the temperature control to 37 degrees Celsius.
Then open the gas control and set the carbon dioxide to 5%When the temperature and gas have reached their targets, open the plate reader drawer and insert a humidity cassette filled with six milliliters of distilled water on each side into the reader. Confirm that the lid and bottom of the cell culture plate are clean and place the plate on the humidity cassette, put the lid back on and close the drawer. Open the Spark Method editor, select the appropriate plate template and select the wells to be monitored during the experiment.
Add a temperature and carbon dioxide control panel and set the panels to 37 degrees Celsius and 5%respectively. Then tick the wait for temperature/gas boxes. Add a kinetic loop panel and select the duration as the loop type.
Set the duration to five minutes and set the interval type of the experiment to not defined, to obtain a continuous reading. Within the kinetic loop, use the drag and drop function to add two fluorescence intensity panels that will be individually set for the pH-sensitive and the pH-insensitive fluorescent dyes. Set the excitation and emission wavelength to 560 and 590 nanometers respectively for the pH-sensitive dye.
And 495 and 520 nanometers respectively for the pH-insensitive dye. Set the number of flashes to 30 and the z-position to 33, 200 for each fluorophore. Set the multiple read per well to user defined, as a three by three by circle with a border of 4, 750 micrometers.
Click start to initiate the background measurement reading and click okay, to confirm that the lid of the humidity cassette is in place. At the end of the measurement, transfer the plate from the plate reader to a tissue culture safety cabinet and add three microliters of freshly prepared dextran-coupled fluorescent dye solution to the apical surface of the cells. Then return the plate to the cell culture incubator overnight.
For the kinetics measurement, open the method file used for the background measurements. When all of the parameters have been set, open the plate reader drawer and insert the humidity cassette. Place the clean plate in the humidity cassette with its cover and click start to initiate the fluorescence readings.
Then click okay, to confirm that the lid of the humidity cassette is in place. After a minimum of 12 cycles, click pause to interrupt the experiment and remove the plate to basolaterally apply any drugs or agonists to the appropriate samples. Return the plate to the humidity cassette on the tray and reposition the humidity cassette lid, before clicking continue, to further record the ASL pH and to monitor the effect of the treatment.
For in situ pH calibration, remove the plate from the plate reader and aspirate the basolateral solution. Add 750 microliters of highly buffered standard curve solution to the basolateral compartment and one microliter of solution to the apical surface. Switch off the carbon dioxide on the plate reader and return the plate to the humidity cassette.
Then set the plate reader with same parameters as demonstrated, but with no carbon dioxide, and start the fluorescence readings every five minutes for one to one and a half hours. For data analysis, select all of the mean data for each sample and, or condition for both wavelengths in the background file and copy and paste the data into a new file. Then calculate the mean background for each well and each wavelength.
After copying and pasting the calibration and kinetic data in the same manner, subtract the background from each data point for each wavelength. For each time point and every sample, calculate the ratio between the pH-sensitive and the pH-insensitive fluorescence. For each time point, generate a standard curve from the ratios and plot the known pH values on the x-axis and the ratios on the y-axis.
Determine the time point at which the ratios are stable, fit a linear regression line and obtain the equation for this line. Then, calculate the pH for each time point and plot the pH on the y-axis and the time on the x-axis. In this representative preliminary experiment without cells at the same pH and same concentration, the pH-sensitive and pH-insensitive emission ratios differed depending on the volume of the dye.
Additionally, at the same pH and the same volume, different dye concentrations provided different ratio values, indicating that changes in the volume or dye concentration affects the absolute value of the pH calculated from the emission ratio. In addition, the time required for temperature equilibration is approximately 15 to 20 minutes regardless of dye volume or concentration. ASL pH values obtained from a single global standard curve demonstrate a significant difference between non-cystic fibrosis and cystic fibrosis cultures.
Whereas, the ASL pH is not significantly different between cystic fibrosis and non-cystic fibrosis human airway epithelial cells when the pH is calculated from independent standard curves. Therefore, it is important to generate independent calibration curves for each experiment and within each experiment, for each donor sample, as when the calibration curves are averaged together, higher pH-sensitive, pH-insensitive ratio values are found in cystic fibrosis cultures indicating a more acidic pH. As expected, the addition of basolateral forskolin significantly increases the ASL pH in non-cystic fibrosis cultures only.
As these cells have been kept under sterile condition, they can be washed, re-fed and used a few days later for future experiments. All biological human specimens should be regarded as potentially hazardous. So make sure to use the appropriate level of confinement and personal protective equipment depending on your samples.
It's important to perform the background measurements and the pH calibrations for each set of samples in order to reduce inter-donor variability and improve accuracy.
