Selection of fluorescent ion indicators for simultaneous measurements of pH and Ca2+

The advent of fluorescent ion sensitive indicators has improved our understanding of the mechanisms involved in regulating pH(i) and [Ca2+](i) homeostasis in living cells. However, changes in [Ca2+](i) can alter pH(i) regulatory mechanisms and vice versa, making assignment of either ion to a particular physiological response complex. A further complication is that all fluorescent Ca2+ indicators are sensitive to protons. Therefore, techniques to simultaneously measure these two ions have been developed. Although several combinations of pH and Ca2+ probes have been used, few systematic studies have been performed to assess the validity of such measurements. In vitro analysis (i.e. free acid forms of dyes) indicated that significant quenching effects occurred when using specific dye combinations. Fura-2/SNARF-1 and MagFura-2/SNARF-1 probe combinations were found to provide the most accurate pH and [Ca2+] measurements relative to Fluo-3/SNARF-1, Ca2+-Green-1/SNARF-1, or BCECF/SNARF-1. Similar conclusions were reached when probes were calibrated after loading into cells. The magnitude of interactions between pH and Ca2+ probes could be a factor which may limit the use of certain specific combinations. Loading of probes that exhibit interactions into distinct intracellular compartments (i.e. separated by a biological membrane) abolished the quenching effects. These data indicate that interactions between the probes used to simultaneously monitor pH and Ca2+ must be considered whenever probe combinations are used.