Figure 5.

Measurement of intraorganellar pH in HAP1 cells using the ratiometric dye LysoSensor™ Yellow/Blue DND-160 and a microplate reader. HAP1 cells were treated as described in the stepwise procedures with the caveat that, to determine the effects of long-term treatment, cells were incubated with LysoSensor™ Yellow/Blue DND-160 (1 μM) over a time course spanning 1 min to 2 h in generating the pH calibration curves and in acquiring experimental data. Cells were subsequently analyzed in triplicate using a microplate reader and SoftMax® Pro V5 software to determine fluorescence intensity ratios. Emissions were collected at 440 and 540 nm for excitations at 329 and 380 nm, respectively. Graphs are shown of the pH calibration curves generated based on fluorescence intensity measurements of HAP1 cells incubated with pH calibration curve buffers of the indicated pH-values and analyzed using a microplate reader. The pH calibration curves were generated in parallel with acquiring experimental data. The final graph shows the average pH of all labeled acidic organelles combined at various time points of LysoSensor™ Yellow/Blue DND-160 incubation in HAP1 cells, as determined by using microplate reader-based ratiometric measurement. Results from incubation of HAP1 cells with this weak-base dye over a time course are indicative of the rapid alkalinizing effect LysoSensor™ Yellow/Blue DND-160 can have on intraorganellar pH. Fluorescence intensity ratios were converted to pH-values by fitting of data to the corresponding pH calibration curves. For HAP1 cells, the average intraorganellar pH of all LysoSensor™ Yellow/Blue DND-160-labeled organelles combined was calculated at the following values for the respective time points: 4.97 ± 0.08 (1 min), 4.89 ± 0.08 (5 min), 5.4 ± 0.1 (20 min), 5.15 ± 0.04 (30 min), and 5.72 ± 0.02 (2 h). Data are presented as the average ± SEM.