Flow cytometric calibration of intracellular pH measurements in viable cells using mixtures of weak acids and bases.

We describe a new method for calibrating intracellular pH (pH1) measurements by flow cytometry, based on the null point method proposed originally by Eisner et al. (Pflügers Arch 413:553-558, 1989). The method involves suspending cells loaded with pH-sensitive dyes, such as SNARF-1 or BCECF, in defined mixtures of the weak acid butyric acid and the weak base trimethylamine. Only the uncharged forms of these agents freely permeate the plasma membrane. The weak acid donates protons intracellularly, whereas the weak base accepts them. In accordance with the Henderson-Hasselbalch equation, when cells are exposed to these mixtures, the steady-state pHi is displaced, and the fluorescence signal reflects this new pHi. The null point method described by Eisner et al. derives pHi by determining the molar ratio of acid to base that produces no change in fluorescence signal. In this paper, we show that it is not necessary to obtain the true null point, because a calibration curve can be derived from "pseudo null" values whose pHi is defined by the equation pHi = pHe -0.5 log [(AT)/(BT)], where pHe is the extracellular pH, and (AT) and (BT) are the total concentrations of weak acid and base in the suspension. We refer to this as the "pseudo null calibration method." It is rapid, technically simple, and reproducible. Compared with the widely used nigericin calibration method, it is not influenced by the intracellular potassium concentration; therefore, it may give a more reliable estimate of the absolute value of pHi.