Intracellularly trapped pH indicators.

The trapped indicator technique has several advantages for monitoring intracellular pH. It can be performed with equipment available in most laboratories, using either fluorescence or absorbance measurements. It is nondestructive to cells, and fluorescein dyes seem to have little effect on cell metabolism (however, see Spray et al., 1984). Continuous real-time monitoring of intracellular pH is possible, and the method can detect changes of 0.01 pH. It is sensitive enough for studies with monolayers (Thomas et al., 1982) or individual cells (Slavik and Kotyk, 1984; Udkoff and Norman, 1979). In addition, pH changes in the cytoplasmic and mitochondrial compartments can be distinguished by judicious use of CF and F. On the negative side, one of the main problems encountered is that of leakage, especially for F. Spectral measurements must be corrected for leakage in order to assess pH accurately. Three ways of minimizing leakage are as follows: (1) Use a less leaky indicator, such as BCECF (Rink et al., 1982); (2) lower the incubation temperature; (3) continuously remove external indicator by perfusion technique (Boron, 1982). Although CFA2 specifically monitors cytoplasmic pH in several different cell types, this may not necessarily be a general phenomenon. As shown in this chapter, it can report mitochondrial pH transitions if it is first hydrolyzed by mitochondrial esterases. Thus, this specificity for the cytoplasm should be established for any new cell type studied. As a final note, Spray et al. (1984) have recently reported that the intracellular hydrolysis of certain membrane-permeant esters causes an acidification of the cytoplasm in several cell types. The acidification was considerably in excess of that expected from the small amount of acid generation caused by the esterase reaction. How general this phenomenon is remains to be established.