A novel method for absolute calibration of intracellular pH indicators

In this paper we present methods to measure intracellular pH (pHi) with fluorescent indicators. These methods are based on the change in intracellular pH following the addition of weak acids and weak bases to the extracellular medium. The first method requires that the fluorescence of the indicator is proportional to the change in pHi that follows the addition of a weak acid or weak base to the extra-cellular medium. The second is a null method which uses a mixture of weak acid and weak base that does not change the fluorescent signal. This null method can be used in situations in which the fluorescent signal is a monotonic but non-linear function of pH. The first method depends upon four assumptions. (i) That only the uncharged forms of the weak acids and bases cross the surface membrane. (ii) That the pKa is the same inside and outside the cell. (iii) That the buffering power is constant. (iv) That there is no significant pH regulation on the time scale of the change in pHi. The null method only requires the first two assumptions. We have made estimates of pHi in four different cell types and compared the results obtained with these methods with those obtained from other methods of pHi calibration.