Lysosomal pH measurement

Quantification of lysosomal pH was performed using a ratiometric lysosomal pH dye LysoSensor^™ Green DND-189 (Thermo Scientific, San Jose, CA, USA). Cells were seeded on the 35 mm confocal dishes and assigned into calibration curve groups (pH = 4.5, 5, 5.5, 6, 6.5, 7) or experimental groups. After treatment, cells were labeled with 1 µM LysoSensor^™ Green DND-189 for 1 h at 37 °C in regular medium. Excessive dye was then washed out using PBS. For experimental groups, cells are ready for fluorescence measurements under a confocal microscope (LSM 800, Carl Zeiss, Jena, Germany). For calibration curve groups, cells were treated for 10 min with 10 µM monensin and 10 µM nigericin in MES buffer with various pH from 4.5 to 7. The MES buffer consisted of 5 mM glucose, 20 mM MES, 1 mM CaCI₂, 1 mM MgCI₂, 130 mM NaCl, and 10 mM KCI. The pH of the MES buffer was adjusted by NaOH or HCl. At least 40 cells from each group were imaged by a confocal microscope. The integrated density of pH-dependent fluorescence was measured by Image J software. The pH calibration curve was generated from the plot using Microsoft Excel. The pH of the experimental groups was calculated according to the fluorescence density and calibration curve.