Filter based imaging system

Endolysosome pH was measured ratiometrically using LysoSensor dye (DND-160, Invitrogen), a dual excitation dye for pH measurements of acidic organelles. Cells were treated with LysoSensor (3 μM) for 3 min at 37°C. Light emitted at 510 nm in response to excitation at 340 and 380 nm was measured for 20 ms every 5 s using a filter-based imaging system (Zeiss) and data were analyzed using Slidebook 6 software (3i, Denver). 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 and 7.0 with HCl/NaOH.^{52 (link)–54 (link)}

As previously described (4 (link)–6 (link)),
endolysosome pH was measured using a ratiometric indicator-dye, LysoSensor Yellow/Blue DND-160; a dual excitation dye that allows
for the measurement of acidic organelles independent of dye loading efficiency. U87MG cells were loaded with 10 μM DND-160
for 5 minutes at 37°C. Post-incubation, dye-containing media was removed and PBS was added to the cells prior to them being
taken for imaging. CellLight Lysosome-RFP (Thermosfisher), incubated in cells overnight, was used to label lysosomes in
combination with LysoSensor DND-160 for selectivity of lysosomal labeling. Light emitted at 520 nm in response to excitation at
340 nm and 380 nm was measured for 2 msec every 10 seconds using a filter-based imaging system (Zeiss, Germany). The ratios of
light excited (340/380 nm) versus light emitted (520 nm) were converted to pH using a calibration curve established with 10
μM of the H⁺/Na⁺ ionophore monensin, and 20 μM of the H⁺/K⁺ ionophore
nigericin; both were dissolved in 20 mM 2-(N-morpholino) ethane sulfonic acid (MES), 110 mM KCl, and 20 mM NaCl adjusted to pH 3.0
to 7.0 with HCl/NaOH and the linear range is 4.5 to 6.0 pH.

As previously described (Liu et al. 2008 (link), Hui et al. 2012a (link)), endolysosome pH was measured using a ratio-metric endolysosome pH indicator dye (LysoSensor Yellow/Blue DND-160, ThermoFisher Scientific, Euegene, US); a dual excitation dye that permits pH measurements in acidic organelles independently of dye concentration. Neurons were loaded with 2 µM LysoSensor for 5 minutes at 37°C. Light emitted at 520 nm in response to excitation at 340 nm and 380 nm was measured for 20 msec every 30 seconds using a filter-based imaging system (Zeiss, Germany). The ratios of light excited (340/380 nm) versus light emitted (520 nm) were 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; both were dissolved in 20 mM 2-(N-morpholino) ethane sulfonic acid (MES), 110 mM KCl, and 20 mM NaCl adjusted to pH 3.0 to 7.0 with HCl/NaOH.