Calcium indicators and ionophores

Aladdin helps you find the best indicators for imaging Ca2+ and cellular function and the ionosphere.

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

Using this guide, you can find the best Ca2+ indicator, chelator and ion mass for your experiments.

Ca2+ indicators at a glance

Indicator	Excitation (nm)	Emission (nm)	Kd (nM)
Fura-2	340/380	505	145
Indo-1	346	475/405	230
Fluo-4	488	520	345
Fluo-8	490	520	390
Fluo-5	494	516	2300
Rhod-2	552	581	570

BAPTA

BAPTA, a non-fluorescent Ca2+ chelator, was extracted from EGTA and was 105 times more selective for Ca2+ than Mg2+. It is commonly used for buffering and has a Kd of 160 nM in the absence of Mg2+. Also available in AM form.

Fura-2

Fura-2 is the first commercially available fluorescent calcium indicator. The excitation ratio was 340 nm/380 nm, and the emission wavelength was 505 nm. There are thousands of references to Fura-2 in the scientific literature and it is by far one of the most important Ca²⁺ indicator dyes.

Indo-1

Indo-1 was introduced at the same time as Fura-2 in 1986. It's also proportional, but in a different pattern. When excited at 346 nm, it exhibits an emission ratio at 475 nm/405 nm, but not an excitation ratio. Unlike Fura-2, it has a tendency to photobleach. It has also been used in a wide variety of applications, exemplified by a large number of literature publications.

Fluo-8

Since its introduction in 1989, fluorescence imaging has revealed the spatial dynamics of many fundamental processes in the Ca²⁺ signaling pathway. Fluo-8(or Fluo-2 medium affinity) was found to be brighter (1.5 times) than Fluo-4 in cell experiments. It improves cell loading and Ca²⁺ response while maintaining convenient Fluo-3 and Fluo-4 spectral wavelengths with maximum excitation at 490 nm and maximum emission at 520 nm. Fluo-8 loading can be performed at room temperature.

Rhod-2

Rhod-2 was first developed in 1989, with fluorescence excitation and emission extremes at 552 nm and 581 nm, respectively. Before Ca²⁺ binding, Rhod-2 is essentially nonfluorescent and becomes more fluorescent with increasing Ca²⁺ concentration. The longer excitation and emission of Rhod-2 make this indicator very useful in experiments with cells and tissues with high levels of autofluorescence and for multiplexing with other fluorescent dyes with shorter wavelengths. The AM ester forms of these rodamine-based indicators are cationic, which can result in potential-driven uptake into mitochondria.

Fluo-4 AM

Fluo-4 is an analogue of Fluo-3 in which two chlorine substituents are replaced by fluorine, which results in enhanced fluorescence excitation at 488 nm and therefore higher fluorescence signal levels.

Fluo-5 AM

Fluo-5 AM is a Fluo-4 analogue but has a reduced calcium binding affinity and is therefore suitable for detecting intracellular calcium levels, which saturate the Fluo-4 AM indicator.

Ca2+  ionophores

A23187(calcin) has an intrinsic fluorescence that can be excited by UV light, making it less useful for Ca2+ indicators that can be excited by UV light, such as Fura-2, but still useful for long-wavelength Ca2+ indicators, such as Fluo-2.

5-bromine A-23187 is non-fluorescent and therefore compatible with all Ca2+ indicators. It is commonly used for in situ calibration of fluorescent Ca2+ indices to balance intracellular and extracellular Ca2+ concentrations and to allow Mn2+ entry into the cell to quench dye fluorescence in the cell.

Ionomycin is an effective Ca2+ ion cell body, which is commonly used to calibrate fluorescence Ca2+ index and modify intracellular Ca2+ concentration, and to study the regulatory characteristics of Ca2+ in cellular processes.

Reference

1.Erdahl WL, Chapman CJ, Taylor RW, Pfeiffer DR. Ca2+ transport properties of ionophores A23187, ionomycin, and 4-BrA23187 in a well defined model system. Biophys J. 1994 May;66(5):1678-93. 
2. Drummond IA, Lee AS, Resendez E Jr, Steinhardt RA. Depletion of intracellular calcium stores by calcium ionophore A23187 induces the genes for glucose-regulated proteins in hamster fibroblasts. J Biol Chem. 1987 Sep 15;262(26):12801-5.