Steady-state lysosomal pH in macrophages. (A) The lysosomes of murine macrophages were loaded with pH-sensitive dextrans and their pH measured by ratiometric imaging. A representative field of RAW264.7 macrophages is shown in A (bar, 10 µm) with the corresponding differential interference contrast (DIC) image in the inset. At the end of each experiment, an in situ calibration was performed as described in Materials and methods to generate a curve relating the 490 nm/440 nm excitation ratio to pH (B). (C) The macrophage cell lines RAW264.7 (black bars) and J774 (gray bars) were either left untreated (5 experiments with a total of 669 lysosomes, and 5 experiments, 1103 lysosomes, respectively) or treated with 5 µM (3 experiments, 538 lysosomes; and 4 experiments, 810 lysosomes, respectively) or 10 µM (3 experiments, 304 lysosomes; and 4 experiments, 861 lysosomes, respectively) of the CFTR inhibitor CFTR_INH-172 for 1 h, and the lysosomal pH was recorded. In both cell lines, treatment with 500 nM CcA for 1 h dissipated the lysosomal pH to values greater than 6.5, the upper limit of sensitivity of the pH probe. (D) Lysosomal pH in alveolar macrophages harvested from wild-type or cftr^−/− mice (5 experiments, 509 lysosomes; and 5 experiments, 395 lysosomes, respectively). Wild-type cells were also incubated for 1 h in the presence of 10 µM of CFTR_INH-172 (4 experiments, 450 lysosomes). (E) To determine whether CFTR functions as the counter-ion conductance for the proton leak in lysosomes, wild-type (3 experiments, 24 lysosomes) and cftr^−/− (4 experiments, 34 lysosomes) alveolar macrophages were treated with 1 µM CcA and the rate of alkalinization was measured over the first 5 min.