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 (pH(i)) meas urements by flow cytometry, based on the null point method proposed or iginally by Eisner et al. (Pflugers Arch 413:553-558, 1989). The metho d involves suspending cells loaded with pH-sensitive dyes, such as SNA RF-1 or BCECF, in defined mixtures of the weak acid butyric acid and t he weak base trimethylamine. Only the uncharged ferns of these agents freely permeate the plasma membrane. The weak acid donates protons int racellularly, whereas the weak base accepts them. In accordance with t he Henderson-Hasselbalch equation, when cells are exposed to these mix tures, the steady-state pH(i) is displaced, and the fluorescence signa l reflects this new pH(i). The null point method described by Eisner e t al. derives pH(i) by determining the molar ratio of acid to base tha t produces no change in fluorescence signal. In this paper, we show th at it is not necessary to obtain the true null point, because a calibr ation curve can be derived from ''pseudo null'' values whose pH(i) is defined by the equation pH(i) = pH(e) - 0.5 log [(A(T))/(B-T)], where pH(e) is the extracellular pH, and (A(T)) and (B-T) are the total conc entrations of weak acid and base in the suspension. We refer to this a s the ''pseudo null calibration method.'' It is rapid, technically sim ple, and reproducible. Compared with the widely used nigericin calibra tion method, it is not influenced by the intracellular potassium conce ntration; therefore, it may give a more reliable estimate of the absol ute value of pH(i). (C) 1996 Wiley-Liss, Inc.