Calcium indicators and ionophores

Find the best indicator and ionophore for imaging Ca²⁺ and cell function.

Calcium (Ca²⁺) is an important ubiquitous second messenger, involved in the regulation of a diverse range of cellular processes, including cell proliferation, gene transcription, muscle contraction, and endocytosis. 

Use this guide to find the optimal Ca²⁺ indicator, chelators, and ionophores for your experiments.

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Ca²⁺ indicators at a glance

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BAPTA

BAPTA is a non-fluorescent Ca²⁺ chelator which was derived from EGTA to give 105-fold selectivity for Ca²⁺ over Mg²⁺. It is commonly used in buffering and has a K_d of 160 nM in the absence of Mg²⁺. Also available in AM form.

Fura-2

Fura-2 is one of the first commercially available fluorescent calcium indicators. It exhibits excitation ratiometry at 340 nm/380 nm with emission at 505 nm. There are thousands of Fura-2 references in the scientific literature and it is one of most important Ca²⁺ indicator dyes to-date.

Indo-1

Indo-1 was introduced at the same time as Fura-2 in 1986. It is also ratiometric, but different in mode. Rather than being excitation ratiometric, it exhibits an emission ratio at 475 nm/405 nm when excited at 346 nm.  Unlike Fura-2, it has a tendency to photobleach.  It has also been used in a variety of applications, exemplified by the large number of literature publications.

Fluo-8

Since its introduction in 1989, Fluo imaging has revealed the spatial dynamics of many elementary processes in Ca²⁺ signaling. Fluo-8 (or Fluo-2 Medium Affinity) has been found to be brighter (1.5x) than Fluo-4 in cellular experiments. It offers improved cell loading and Ca²⁺ response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelength of maximum excitation at 490 nm and maximum emission at 520 nm. Fluo-8 loading can be performed at room temperature.

Rhod-2

Rhod-2, with fluorescence excitation maxima at 552 nm and emission maxima at 581 nm, was first introduced in 1989.  Rhod-2 is essentially non-fluorescent before Ca²⁺ binding, becoming more fluorescent with increasing Ca²⁺ concentration. The longer excitations and emissions of Rhod-2 make the indicator useful for experiments in cells and tissues that have high levels of autofluorescence and for multiplexing with other fluorescent dyes of shorter wavelengths. The AM ester forms of these rhodamine-based indicators are cationic, which can result in potential-driven uptake into mitochondria.

Fluo-4 AM

Fluo-4 is an analog of Fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels.

Fluo-5 AM

Fluo-5 AM is an analogue of Fluo-4 with reduced calcium binding affinity, for this reason it is suitable for detecting intracellular calcium levels that would saturate the Fluo-4 AM indicator.

Ca²⁺ ionophores

A23187 (Calcimycin) has an intrinsic fluorescence excitable by UV light, making it less useful with UV-excitable Ca²⁺ indicators, e.g., Fura-2, but it is still useful for long-wavelength Ca²⁺ indicators, e.g., Fluo-2.

4-Bromo A-23187 is nonfluorescent and is thus compatible with all Ca²⁺ indicators. It is commonly used for in situ calibrations of fluorescent Ca²⁺ indicators, to equilibrate intracellular and extracellular Ca²⁺ concentrations and to permit Mn²⁺ to enter the cell to quench intracellular dye fluorescence.

Ionomycin an effective Ca²⁺ ionophore that is commonly used to both calibrate fluorescent Ca²⁺ indicators and to modify intracellular Ca²⁺ concentrations and to when studying the regulatory properties of Ca²⁺ in cellular processes.