AAT Bioquest

Fluorescent Calcium Indicators

Calcium acts as a universal second messenger in a variety of cells. Numerous functions of all types of cells are regulated by Ca2+, thus calcium measurement is critical for various biological investiga-tions. Since the 1920s, scientists have attempted to measure Ca2+, but few were successful due to the limited availability of Ca2+ probes. The first reliable measurement of Ca2+ was performed by Ridgway and Ashley by injecting the photoprotein aequorin into the giant muscle fiber of the barnacle. Subsequently, in the 1980s, Tsien and colleagues produced a variety of fluorescent indicators. Among them Indo-1, Fura-2, Fluo-3 and Rhod-2 have been the most valuable dyes for measuring Ca2+ with a fluorescence instrument. In recent years, AAT Bioquest has introduced the most robust calcium probes: Fluo-8® and Cal-520™, both of which enable the high throughput screening of GPCR and calcium channel drug discovery targets through the convenient calcium detection. FLIPR® and FlexStation® instruments of Molecular Devices, FDSS®/µCELL of Hamamatsu and NOVOstar of BMG Technologies have further accelerated the high throughput measurement of calcium for GPCR and ion channel research.

Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Most of these fluorescent indicators are derivatives of BAPTA chelators that incorporate a PET system responsive to calcium. There are quite a few factors that need be considered when selecting a fluorescent Ca2+ indicator. These include:

  • Spectral Properties: For UV excitation, Indo-1 and Fura-2 are widely used. Fura-8™ is a newly developed excitation-ratioable calcium dye. Its AM is superior to Fura-2 AM with higher signal/background ratio in cells. Fluo-8® and Cal-520™ are preferred for 488 nm excitation while Cal-590™, Cal-630™, Rhod-2 and Rhod-4™ are used for red emissions.
  • Measurement Mode: Ion indicators that exhibit spectral shifts upon ion binding can be used for ratiometric measurements of Ca2+ concentration, which are essentially independent of uneven dye loading, cell thickness, photobleaching effects and dye leakage. Excitation and emission wavelength preferences depend on the type of instrumentation being used, as well as on sample autofluorescence and on the presence of other fluorescent or photoactivatable probes in the experiment. Indo-1, Fura-2 and our newly developed Fura-8™ are primary choices for ratiometric measurements while Fluo-3, Fluo-4, Fluo-8®, Cal-520™, Cal-590™, Cal-630™, Rhod-2 and Rhod-4™ are predominantly used for single wavelength measurements.
  • Permeability of Ca2+ Indicators (salt or AM ester): The salt forms are typically loaded into cells by microinjection, microprojectile bombardment or electroporation, or used for extracellular assays. In contrast, the cell-permeant acetoxymethyl (AM) esters can be passively loaded into cells, where they are cleaved to cell-impermeant products by intracellular esterases.
  • Dissociation Constant (Kd): The desired indicators must have a proper Kd compatible with the Ca2+ concentration range of interest. The Kd values of Ca2+ indicators are dependent on many factors, including pH, temperature, ionic strength, viscosity, protein binding, the presence of Mg2+ and other ions. Consequently, Kd values for intracellular indicators are usually significantly higher than the corresponding values measured in cell-free solutions.

Among the visible light-excitable calcium indicators, Fluo-8®, Fluo-4, Fluo-3, Rhod-2 and Rhod-4™ are most commonly used. Fluo-8® indicators are widely used in flow cytometry and confocal laser-scanning microscopy. More recently, Fluo-8® AM has been extensively used for high throughput screening GPCR targets. Fluo-8® is essentially nonfluorescent unless bound to Ca2+ and exhibits a quantum yield of ~0.15 in the presence of saturating Ca2+ and a Kd of 390 nM for Ca2+. Cal-520™ is by far the best 488 nm-excitable green fluorescent calcium indicator with a significantly improved signal/background ratio and intracellular retention.

The long-wavelength Rhod-4™ is a valuable alternative Ca2+ indicator to the green fluorescent Fluo-8®, Fluo-4 and Fluo-3 for experiments in cells and tissues that have high levels of autofluo-rescence. Rhod-5N has a lower binding affinity for Ca2+ than any other BAPTA-based indicator (Kd = ~320 µM) and is suitable for Ca2+ measurements from 10 µM to 1 mM. Like the parent Rhod-2 indicator, Rhod-5N is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca2+. Both Fluo and Rhod indicators are available as cell-impermeant potassium salts or as cell-permeant AM esters.

Table 1. Classic Single Wavelength Fluorescent Calcium Indicators

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
20500Cal Green™ 1, hexapotassium salt506531190 nM 10x50 ug
20501Cal Green™ 1, AM [Equivalent to Calcium Green-1, AM]506531190 nM 10x50 ug
21011Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*506526390 nM 1 mg
21018Fluo-3, pentaammonium salt506526390 nM 1 mg
21017Fluo-3, pentapotassium salt506526390 nM 1 mg
21016Fluo-3, pentasodium salt506526390 nM 1 mg
21064Rhod-2, AM *UltraPure Grade* *CAS#: 145037-81-6*549578570 nM 20x50 ug
21067Rhod-2, tripotassium salt549578570 nM 1 mg
21068Rhod-2, trisodium salt549578570 nM 1 mg
21070Rhod-5N, AM5515770.3 mM 1 mg
21072 Rhod-5N, tripotassium salt5515770.3 mM 1 mg