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

Abstract

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.

Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells

“…Several calibration methods based on addition of solutions of a weak acid and a weak base have been proposed. These include the method of Szatkowski and Thomas (5), the null point method proposed by Eisner et al (6), and the pseudo‐null point method proposed by Chow et al (7). Szatkowski and Thomas (5), derived the following equation for change in pH i as a function of pH e : where [A] and [B] are the concentrations of weak acid and weak base, respectively, and ΔpH a and ΔpH b are the magnitudes of changes in pH with the addition of weak acid.…”

“…Eisner et al (6) extended this method to include the use of a spectrofluorometer and fluorescent indicator in place of the pH‐sensitive microelectrodes used by Szatkowski and Thomas (5). Chow et al (7) further developed this approach by using flow cytometry and the fluorescent indicator SNARF.…”

“…The null point method (method 3) requires titration of acid and base to determine the concentrations of acid and base required to achieve no change in fluorescence ratio. Chow et al (7) described a calibration method, based on the null point method, that eliminates the need for time‐consuming titration. In this method (the pseudo‐null calibration method), a series of predetermined calibration solutions is used (Table 1) (7).…”

“…Chow et al (7) described a calibration method, based on the null point method, that eliminates the need for time‐consuming titration. In this method (the pseudo‐null calibration method), a series of predetermined calibration solutions is used (Table 1) (7). By using these solutions, the pseudo‐null method of calibration obviates a perfusion system, thereby allowing other methods for measuring fluorescence to be used in conjunction with the null point hypothesis, e.g., flow cytometry.…”

“…Pseudo‐null point solutions were made according to the method of Chow et al (7). The method can be briefly summarised as follows: six times concentrated standard solution was made by adding 1 M butyric acid and 1 M trimethylamine to the basal solution (pH e 7.4); the basal solution was solution 3, growth medium, or solution 4 (Table 2).…”