ASL pH measurements

Fully differentiated ALI cultures were derived from wild-type, CFTR-KO (ref. ³⁶) and FOXI1-KO primary tracheal basal cells. ASL pH was measured with slight modifications to that previously described¹⁵. In brief, the ratiometric pH indicator SNARF-conjugated dextran dye (ThermoFisher Scientific) was used to generate pH standard curves and directly measure apical pH on differentiated ALI cultures. ALI cultures were maintained in basolateral HCO₃⁻-containing buffer and the microscope chamber was humidified in a 5% CO₂ atmosphere at 37 °C. SNARF-conjugated dextran powder was directly applied to the apical surface through a 5 µm mesh. A confocal microscope (Zeiss LSM 880) was used to excite the SNARF dye at 488 nm and measure fluorescence intensity at 580 nm and 640 nm from 6–8 areas of interest in each ALI culture. Fluorescence ratios were converted into pH values by using the standard curves as previously described⁴³.