Intracellular pH Measurement

Measurements of pHi were performed using the pH-sensitive fluorescent dye 2,7-bis- (2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM, Invitrogen) ¹⁵. In brief, Cells were incubated at room temperature with Ringer solution (154 mM NaCl, 2.2 mM CaCl₂, 5.6 mM KCl, 2.4 mM NaHCO₃, 2 mM Tris-HCl, pH 7.4) containing 2 µM BCECF-AM (Invitrogen) for 10 min. The fluorescence intensity was detected by lympus Provis fluorescence microscope (Nikon Eclipse Ti-SR) in same exposure time and calculated by Image-Pro Plus 6.0 software. pH calibration was performed after each experiment by the nigericin (MedChemExpress) technique. A ten-point in situ pH calibration (6.4 to 8.2) was performed in sodium-free calibration buffer (125mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 20 mM HEPES and 10 µM nigericin).