Experimental lysosomal pH measurements

Lysosomal pH was measured fluorometrically in peritoneal macrophages and macrophage-derived RAW264.7 cells, using methods described previously²⁸. In brief, macrophages were incubated for 40 h with CFZ crystals. 20 h prior to measuring pH, cells were incubated for 15–18 h with 150 µg/ml Oregon Green-labeled dextran-10 kD (OGDx; Thermo-Fisher Scientific), and transferred to unlabeled culture medium for 3–5 h to allow OGDx to traffic fully to lysosomes. Following transfer to Ringer’s buffer, cells on Mat-tek dishes were observed in a Nikon TE300 microscope equipped for multichannel fluorescence microscopy with a 60X objective lens (N.A. 1.4). Phase contrast and three fluorescence images (excitation/emission: 440 nm/535 nm; 485 nm/535 nm; and 580 nm/630 nm) were collected from each field. The 580 nm/630 nm image was used to quantify clofazamine fluorescence. The ratio of the 485 nm/525 nm to 440 nm/525 nm images were used to measure lysosomal pH. Calibration of fluorescence ratios was obtained from cells incubated in ionophores and buffers as described in Davis and Swanson²⁸. Average lysosomal pH was determined for individual cells.