Metamorph system

pH_i was measured with BCECF at dual excitation wavelengths of 495 nm and 440 nm. BCECF fluorescence was read at emission wavelengths above 530 nm. Isolated SMG cells attached onto coverslips were loaded in the chamber with BCECF in the presence of 0.05% Pluronic F-127 for 15 min incubation with PSS at room temperature with 6 μM BCECF-AM. After stabilizing the fluorescence, the cells were perfused with PSS for at least 5 min before measuring pH_i at 37°C. CBE activity was measured by incubating the cells with CO₂-saturated HCO₃⁻-buffered media to acidify the cytosol. CBE activity was initiated by perfusing the cells with Cl⁻-free HCO₃⁻-buffered media containing 140 mM Na⁺. The emitted fluorescence was monitored with a CCD camera (Photometrics, Tucson, AZ) attached to an inverted microscope (Olympus, Japan) and analyzed with a MetaMorph system (Molecular Devices, PA). Fluorescence images were obtained at 1 sec intervals and the background fluorescence was subtracted from the raw background signals at each wavelength. CBE activity was determined from the slopes and the derivatives of the first 30–45 sec of pH_i increases.

The dispersed H1975 cells (5 × 10⁴ cells/well) in Reg, 5 mM Cl⁻ solutions, or RPMI (with 1% FBS each) were added to the upper chamber of a 6-well transwell membrane plate (8.0 μm pore sized insert). The bottom chambers were filled with pH 7.5 or pH 6.5 RPMI (with 10% FBS and 100 U/mL penicillin–streptomycin), followed by the indicated conditions. After incubation for 3 h, the membranes were stained with DAPI (blue) or crystal violet (purple). The membranes were incubated with chilled methanol (preserved at −20 °C) for 1 min at −20 °C to fix the cells and then washed with PBS three times. For DAPI staining, the membranes were incubated with DAPI solution (1μg/mL in distilled water (DW)) for 30 min at 37 °C in the dark and then washed twice with DW. For crystal violet staining, the membranes were incubated with 0.25% crystal violet solution in DW (with 20% methanol) for 10 min at room temperature in the dark and then washed with DW six times. After washing the membranes, the media on the top was carefully removed, and DW was added to the bottom of the plate. The plates were subsequently analyzed using an LSM 700 Zeiss confocal microscope (Carl Zeiss) (for DAPI) or an inverted microscope (Olympus) with Mosaic software (Opto Science, Tokyo, Japan, version 1.6) (for crystal violet). The intensity of the obtained images was measured using the Meta Morph system (Molecular Devices).