Axiovert tv100

The intracellular pH of PANC-1 cells was measured using fluorescent live-cell imaging (Axiovert TV100, Zeiss, Oberkochen, Germany) as described previously [45 (link)]. Cells were loaded with the fluorescent pH indicator 2′7′-bis(carboxyethyl)-5-carboxyfluorescein (BCECF-AM) (3 µM) for up to 2 min. The excitation wavelength alternated between 490 nm and 440 nm. The emitted fluorescence was detected at 510 nm. The mean fluorescence of each cell was measured in 10 s intervals. Data acquisition and the polychromator (Visitron Systems, Puchheim, Germany) were controlled by the program VisiView (Visitron Systems). The cells were superfused with prewarmed (37 °C) CO₂/HCO₃⁻-buffered Ringer’s solution (116 mM NaCl; 24 mM NaHCO₃; 5.4 mM KCl; 0.8 mM MgCl₂; 1.2 mM CaCl₂; 5.5 mM Glucose) at pH 7.4. NaHCO₃ was lowered to 4.7 mM for pH 6.6. pH measurements were calibrated with a two-point calibration (130 mM KCl; 1.2 mM CaCl₂; 0.8 mM MgCl₂; 10 mM Hepes; 5.5 mM Glucose; pH 7.5 and pH 6.5; supplemented with 10 µM Nigericin) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). For data analysis, the mean fluorescence intensity of the cell area was measured and corrected for background fluorescence. Afterward, the 490 nm/440 nm ratio was determined, and the pH_i was calculated with a linear regression of pH 6.5 and 7.5.