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

✍ Scribed by Sue Chow; David Hedley; Ian Tannock

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 621 KB
• Volume: 24
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19960801)24:4<360::aid-cyto7>3.0.co;2-j

✦ Synopsis

We describe a new method for calibrating intracellular pH (pH,) measurements by flow cytometry, based on the null point method proposed originally by Eisner et al. (Pflugers Arch 413:55+558, 1989).

The method involves suspending cells loaded with pH-sensitive dyes, such as SNARF-1 or BCECF, in deh e d mixtures of the weak acid butyric acid and the weak base trimethylambe. Only the uncharged forms of these agents freely permeate the plasma membrane. The weak acid donates protons intra- cellularly, whereas the weak base accepts them. In accordance with the Henderson-Hasselbalch equation, when cells are exposed to these mixtures, the steady-state pH, is displaced, and the fluorescence signal reflects this new pH,. The null point method described by Eisner et al. derives pH, 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 fkom "pseudo null" values whose pH, is defined by the equation pH, = pH, -0.5 log [(AT)/(BT)], where pH, 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 pH,. 0 1996 Wfley-Liss, hc.