Simultaneous analysis of intracellular pH and Ca²⁺ from cell populations.

Although changes in both pH(in) and [Ca(2+)](i) have been observed in response to a variety of agonists, it is not clear whether these ionic events work independently or are coordinated to lead to a specific physiological response. One of the fundamental problems in studying these ionic events is that changes in pH(in) modify Ca(2+) regulatory mechanisms and changes in Ca(2+) may modify pH regulation. It is desirable to use a technique that allows concomitant monitoring of these two ions in cell populations with high time resolution. Furthermore, like many Ca(2+) binding proteins, all Ca(2+)-sensitive fluoroprobes are inherently sensitive to pH owing to competition of H(+) for the Ca(2+)-binding sites. This chapter describes experimental paradigms that provide optimum conditions for simultaneous measurement of pH from the fluorescence emission of snarf-1, and Ca(2+) using fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca(2+) both in vitro and in vivo. Moreover, the ratio of the H(+)-sensitive wavelengths of snarf-1 is unaffected by Ca(2+), or the concomitant presence of fura-2 in cells. Although the fluorescence ratio of fura-2 is insensitive to the presence of snarf-1, it is affected by pH, as indicated above. We describe procedures to correct for this effect and to obtain calibration parameters for fura-2 and snarf-1 required to facilitate analysis of pH and Ca(2+) concentrations within cell populations.