Lysosomal pH Measurement

Lysosomal pH was measured using the commercially available dye LysoSensor^TM Yellow-Blue DND-160 (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. In brief, cells were incubated with 1 µM LysoSensor^TM solved in isotonic solution (20 mM MES, 5 mM KCl, 120 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, 10 mM Glucose, pH 7.4) for 5 min and fluorescence intensities were measured at 330 nm and 385 nm on a TECAN Spark Microplate Reader (Tecan Group, Maennedorf, Switzerland). A calibration curve was established for every individual cell line grown on a black 96-well microtiter plate by incubation of cells for 15 min with isotonic solutions with defined pH values (pH 3.0–5.0). Nigericin (10 µM) and Monensin (10 µM) sodium salt (Sigma-Aldrich, St. Louis, USA) were added to dissipate intracellular pH gradients.