Manipulation of intragranular pH in PC12 cells. PC12 cells expressing CgA-ECFP were used to measure intragranular pH using FLIM. (A) Calibration curve of fluorescence lifetimes of CgA-ECFP in the function of intragranular pH. PC12 cells were incubated in calibration solutions that contain 10 µM nigericin and 140 mM KCl buffered at the indicated pH. Data are means ± SEM (n = 15–42 cells from six independent experiments). (B) Schemes illustrating how nigericin (N), NH₄Cl, or V-ATPase inhibitors affect intragranular pH. (C) Intragranular pH plateau values measured in various conditions. In resting conditions (control [ctrl]) pH was 5.51 ± 0.04 (n = 92 cells). After treatment with 5 µM nigericin (nig), granular pH was 7.15 ± 0.06 (n = 30). Increasing concentrations of NH₄Cl led to increasing granular pH, 6.31 ± 0.03 (n = 23) at 5 mM, 6.58 ± 0.04 (n = 37) at 10 mM, and 6.78 ± 0.04 (n = 46) at 20 mM, an effect that was reversible after washing (W) out NH₄Cl, pH 5.92 ± 0.05 (n = 20). The V-ATPase inhibitors bafilomycin A1 (B) and saliphenylhalamide A (S) at 0.4 µM raised the pH to 6.80 ± 0.05 (n = 13) and 6.21 ± 0.06 (n = 19), respectively. **, P < 0.01; ***, P < 0.001. (D) Time course of granular pH variations after NH₄Cl, bafilomycin A1, or nigericin addition (arrow). Typical experiments are shown that were repeated at least four times.