When Intracellular pH Calibration Buffer Kit is used with pHrodo™ Red AM, pHrodo™ Green AM or BCECF Intracellular pH Indicator When used together, intracellular pH can be quantitatively determined. pHrodo™ Red AM and pHrodo™ Green AM have weak fluorescence at neutral pH but increase as pH decreases. pHrodo™ Red and Green dyes are modified with AM ester groups to produce an uncharged molecule that can penetrate cell membranes. Once inside the cell, the lipophilic blocking group is cleaved by nonspecific esterases, forming compounds that remain in the intracellular space. The fluorescence intensity of the probe is an indicator of intracellular pH. Subsequent use of the Intracellular pH Calibration Buffer Kit allows for quantitative determination of intracellular pH.

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 to help balance pH inside and outside the cell. After using pHrodo™ Red AM or pHrodo™ Green AM and measuring the fluorescence intensity under experimental conditions, the cells were resuspended in a calibration buffer supplemented with valinomycin and nigericin and the fluorescence intensity was measured again. Repeat the experiment using the remaining calibration buffer to create a standard curve that can be used to determine intracellular pH relative to experimental conditions.