Abstract
Background
Two calibration methods have been proposed for determining the relation between the fluorescence ratio of a pH‐sensitive fluorescent indicator and intracellular pH (pH~i~). The first method uses nigericin to clamp pH~i~ to external pH (pH~e~) and the second is the null point method. We compared these different calibration methods, solution conditions, and temperatures by using flow cytometry and the fluorescent dye 1,5‐ (and‐6)‐carboxy seminaphtorhodafluor‐1‐acetoxymethyl ester with an NS0 cell line.
Methods
The nigericin method was performed in glucose solutions supplemented with KCl and 2‐(N‐morpholino)ethane sulphonic acid plus tris(hydroxymethyl)aminomethane (solution 1A), a mixture of K~2~HPO~4~/KH~2~PO~4~ in glucose‐solution supplemented solutions (solution 2A), or bicarbonate buffered growth medium supplemented with K~2~HPO~4~/KH~2~PO~4~ (solution 2B); this allowed a range of pH~e~ values to be used. The effect of temperature (22°C or 37°C) on the nigericin calibration curve was also investigated. The null point method was performed by using a series of solutions with a mixture of weak acid and base with a known pH~i~ response.
Results
Using solution 1A as the calibration solution resulted in acidic values of pH~i~ for cells cultured in medium as compared with the values achieved with solution 2A. Using solution 2B did not affect the calibration curve. For the temperatures considered in this study, there was no affect on the calibration curve, but temperature did affect the pH~i~ value of cells in phosphate buffered saline. The pseudo‐null point method used with flow cytometry resulted in a calibration curve that was significantly different (P < 0.05) from that achieved using the nigericin method.
Conclusions
Our data indicates that the choice of calibration solution can affect the reported pH~i~ value; therefore, careful choice of solution is important. © 2005 Wiley‐Liss, Inc.