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

Thomas, D.; Tovey, S. C.; Collins, T. J.; Bootman, M. D.; Berridge, M. J. and Lipp, P. (2000). A comparison of fluorescent Ca²⁺ indicator properties and their use in measuring elementary and global Ca²⁺ signals. Cell Calcium, 28(4) pp. 213–223.

DOI: https://doi.org/10.1054/ceca.2000.0152

Abstract

Quantifying the magnitude of Ca²⁺ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca²⁺ indicators appropriate for a particular application depends upon several considerations including Ca²⁺ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca²⁺ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon 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 in-situ Ca²⁺ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca²⁺ binding affinities between nuclear 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 susceptible to photobleaching than many of the other Ca²⁺ indicators.

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