SELECTION OF FLUORESCENT ION INDICATORS FOR SIMULTANEOUS MEASUREMENTSOF PH AND CA2+

The advent of fluorescent ion sensitive indicators has improved our un derstanding of the mechanisms involved in regulating pH(i) and [Ca2+]( i) homeostasis in living cells. However, changes in [Ca2+](i) can alte r pH(i) regulatory mechanisms and vice versa, making assignment of eit her ion to a particular physiological response complex. A further comp lication is that all fluorescent Ca2+ indicators are sensitive to prot ons. Therefore, techniques to simultaneously measure these two ions ha ve 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 f orms of dyes) indicated that significant quenching effects occurred wh en 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, o r BCECF/SNARF-1. Similar conclusions were reached when probes were cal ibrated after loading into cells. The magnitude of interactions betwee n pH and Ca2+ probes could be a factor which may limit the use of cert ain specific combinations. Loading of probes that exhibit interactions into distinct intracellular compartments (i.e. separated by a biologi cal membrane) abolished the quenching effects. These data indicate tha t interactions between the probes used to simultaneously monitor pH an d Ca2+ must be considered whenever probe combinations are used.