Ratiometric Lysosomal pH Measurement

Endolysosome pH was measured ratiometrically with LysoSensor (LysoSensor Yellow/Blue DND-160, Invitrogen); a dual excitation dye for pH measurements in acidic organelles^{64 (link)}. Cells were loaded with LysoSensor (2 µM) for 5 minutes at 37 °C. Light emitted at 510 nm in response to excitation at 340 nm and 380 nm was measured for 20 ms every 5 seconds using a filter-based imaging system (Zeiss) and data were analyzed using Slidebook 6 software (3i). The ratio of light excited at 340/380 nm and emitted at 510 nm was converted to pH using a calibration curve established using 10 µM of the H⁺/Na⁺ ionophore monensin, and 20 µM of the H⁺/K⁺ ionophore nigericin dissolved in 20 mM 2-(N-morpholino) ethane sulfonic acid (MES), 110 mM KCl, and 20 mM NaCl; pH was adjusted between 3.0 to 7.0 with HCl/NaOH.