How do you use PDMPO to monitor lysosomal pH?

PDMPO (Cat #21204) is a ratiometric imaging probe used to measure the pH of acidic organelles (e.g. lysosomes). It has an excitation maximum at ~360 nm and emission maxima at 450 nm and 550 nm. The ratio of 550/450 increases when pH decreases.

Here is an example protocol for a PDMPO assay:

1. Make Standard pH Calibration Buffer (for example MES Buffer) (pH=4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5)
2. Add 10uM Nigericin and 10uM Valinomycin to the calibration buffer.
3. Incubate Cells with PDMPO for 30min at 37oC.
4. Wash cells once with desired buffer.
5. Incubate cells with pH calibration buffer for 5min.
6. Analyze cells using the appropriate Ex/Em Filters.