Creative Biolabs now offers a broad range of Calcium indicators. Our compounds vary in cell-permeability, structure, and specificity, allowing the effects of calcium to be studied in multiple physiological systems. Our popular products are typically available for immediate shipment.
Ca2+ is an important ubiquitous second messenger, involved in the regulation of a diverse range of cellular processes, including gene transcription, cell proliferation, endocytosis, and muscle contraction. It is essential to quantitatively monitor Ca2+ concentration when study the roles of this important intracellular messenger in cells. The most widely used approach of Ca2+ detection is by the use of fluorescent Ca2+ indicators. Ca2+ indicators are fluorophores coupled to a BAPTA calcium chelator structure. Binding of Ca2+ to the chelator portion of the molecule causes a spectral shift in the dye fluorescence and/or increased dye fluorescence.
Calcium Indicators at a Glance
|Indicator||Excitation (nm)||Emission (nm)||Kd (nM)|
Fura-2 is one of the first commercially available fluorescent calcium indicators. Fura-2 exhibits excitation radiometry at 340 nm/380 nm with emission at 505 nm.
Rather than being excitation ratiometric, Indo-1 exhibits an emission ratio at 475 nm/405 nm when excited at 346 nm. Different from Fura-2, Indo-1 has a tendency to photobleach.
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-8 has been found to be brighter 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.
A Fluo-4 analogue with lower affinity for calcium for detecting intracellular calcium levels.
Rhod-2 was first introduced in 1989 with fluorescence excitation maxima at 552 nm and emission maxima at 581 nm. 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.
Ratiometric and Non-Ratiometric Indicators
- Upon binding to Ca2+, ratiometric indicators undergo a shift either emission wavelength, excitation wavelength, or both. Ratiometric measurements may require specialized imaging setups or filter cubes. Ratiometric readout minimizes the effects of photobleaching, leakage, uneven loading, and varying cell thicknesses in mixed populations, delivering more robust and reproducible results. Besides, ratiometric calcium indicators reduce the problems associated with measuring Ca2+ in cells of unequal thickness. Fura-2 and Indo-1 are typically used to measure changes in calcium concentration by either monitoring excitation or emission, respectively.
- Non-ratiometric indicators show increased fluorescence intensity upon binding to calcium, with no shift in excitation/emission wavelengths.
Salt Form and AM Ester Form
- In salt forms, indicators are water-soluble and membrane impermeant, so loading them into cells requires microinjection.
- AM esters are membrane-permeant forms of indicators that themselves do not bind ions. However, once they have entered cells, they are hydrolyzed by esterases to release the parent ion indicator. Thus, cells or tissues can be loaded with indicator by simply incubating them with a buffer containing the AM ester form.
How to Find the Optimal Calcium Indicator for your Experiments?
You should select a Ca2+ indicator that best suits your needs in consideration of the instrument settings, biological system, and other fluorescent probes used in your experiment. The Kd values give an estimate of the Ca2+ concentration response, usually 0.1-10 Kd, with the caveat that Kd values measured in vitro may differ considerably from those in cells due to differences in ionic strength, pH, viscosity, and Ca2+ buffering by lipids and proteins.
Creative Biolabs has developed a number of calcium indicators to track calcium with intense fluorescent signals and a range of wavelength options. A variety of indicators are available that vary in their Ca2+ Kd or Ca2+ response range, excitation/emission wavelengths, spectral shift, and relative fluorescent quantum yields. For any interested products, please feel free to contact us for more information.