Molecular Probes™ Intracellular pH Calibration Buffer Kit

The Intracellular pH Calibration Buffer Kit enables the quantitation of intracellular pH when used in conjunction with pHrodo™ Red AM, pHrodo™ Green AM, or BCECF intracellular pH indicators. pHrodo™ Red AM and pHrodo™ Green AM are weakly fluorescent at neutral pH but increasingly fluorescent as the pH drops. Modification of pHrodo™ red and green dyes with AM ester groups results in an uncharged molecule that can permeate cell membranes. Once inside the cell, the lipophilic blocking groups are cleaved by nonspecific esterases, resulting in a compound that is retained within the intracellular space. Fluorescence intensity of the probes is then an indicator of intracellular pH. Subsequent use of the Intracellular pH Calibration Buffer Kit allows this intracellular pH to be quantified.

The Intracellular pH Calibration Buffer Kit contains a range of pH calibration buffers (pH 4.5, 5.5, 6.5, and 7.5), as well as valinomycin and nigericin, which help equilibrate the pH inside and outside of cells. After using pHrodo™ Red AM or pHrodo™ Green AM and measuring fluorescence intensity under experimental conditions, the cells are then resuspended in one of the calibration buffers to which valinomycin and nigericin have been added, and the fluorescence intensity measured again. This is repeated with the remaining calibration buffers so that a standard curve can be created and used to pinpoint the intracellular pH associated with the experimental conditions.