A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals

Quantifying the magnitude of Ca2+ signals from changes in the emission of f luorescent indicators relies on assumptions about the indicator behaviour i n situ. Factors such as osmolarity, pH, ionic strength and protein environm ent can affect indicator properties making it advantageous to calibrate ind icators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon sever al considerations including Ca2+ binding affinity, dynamic range and ease o f loading. These factors are usually best determined empirically, This stud y describes the in-situ calibration of a number of frequently used fluoresc ent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Orego n Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high- amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower i n-situ Ca2+ binding affinities than suggested by previously published in-vi tro determinations. Furthermore, for some of the indicators, there were sig nificant differences in the apparent Ca2+ binding affinities between nuclea r and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptibl e to photo- bleaching than many of the other Ca2+ indicators. (C) 2000 Harc ourt Publishers Ltd.