Proprietary, pH-sensitive Molecular Probes® pHrodo™ dyes are almost non-fluorescent at neutral pH and fluoresce brightly in acidic environments, making them ideal for use as pH indicators for a variety of applications. With both red and green pHrodo™ dye choices, you have more opportunities for multiplexing pH determination with other cellular measurements.
pHrodo™ dyes can be used to measure intracellular pH in either the cytosol or in cellular compartments. The minimal dye fluorescence at neutral pH also eliminates the need for wash steps and quencher dyes because any non-internalized dye is essentially nonfluorescent in the culture medium.
Measuring intracellular pH using pHrodo™ indicators
Murine J774A.1 macrophages were labeled with pHrodo™ Green AM Intracellular pH Indicator and pHrodo™ Red 10,000 MW Dextran. The pHi was clamped to the indicated values using the Intracellular pH Calibration Buffer Kit.
pHrodo™ dye is a novel, fluorogenic dye that dramatically increases in fluorescence as the pH of its surroundings becomes more acidic (Figure 5).
The optimal absorption and fluorescence emission maxima of the pHrodo™ Green dye and its conjugates are approximately 509 nm and 533 nm, respectively, while pHrodo™ Red excites at 560 nm and emits at 585 nm. Both pHrodo™ Green and pHrodo™ Red can also be excited with the 488 nm argon-ion laser installed on most flow cytometers, microscopes, and microplate readers.
||pHrodo™ red (left) and pHrodo™ Green (right) dye fluorescence emission spectra. The fluorescence emission spectra of pHrodo™ dye–labeled E. coli were measured in a series of 50 mM potassium phosphate buffers ranging in pH from 4 to 10. The E. coli were at a concentration of 0.1 mg/mL, and the readings were made on a Hitachi F4500 fluorometer, using an excitation wavelength of 532 nm.