Intracellular Ions

The steady-state maintenance of disproportionate concentrations of inorganic cations and anions is a feature of living cells. Homeostatic regulation of these ionic gradients across different compartments is critical for most cellular functions. Measuring concentrations of these ions with both spatial and temporal resolution has become very critical in understanding the physiology of the cells in the research community. Ion probes provide a means of correlating ion channel activation with subsequent changes in intracellular ion concentration. Flux measured by these type of probes also reflects the cells’ membrane potential. We offer many ion probes which effectively measures respected ion efficiently.

Calcium Ion Detection

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 investigations. 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⁺. In recent years, AAT Bioquest has introduced the most robust calcium probes: Fluo-8^®, Cal-520^® and Calbryte™ 520 all of which enable high throughput screening of GPCR and calcium channel drug discovery targets by monitoring calcium flux. FLIPR^® and FlexStation^® instruments of Molecular Devices, FDSS^®/?Cell of Hamamatsu and NOVOstar of BMG Technologies have further accelerated the high throughput measurement of calcium for GPCR and ion channel research.

Fluorescent Single-Wavelength Calcium Indicators

Among 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, confocal laser-scanning microscopy and for high throughput screening of 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™, Cal-590™ and Cal-630™ are valuable alternative Ca²⁺ indicators to the green fluorescent Fluo-8®, Fluo-4 and Fluo-3 for experiments in cells and tissues that have high levels of autofluorescence. 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⁺. All Fluo, Cal and Rhod indicators are available as cell-impermeant potassium salts or as cell-permeant AM esters.

Superior Single-Wavelength Calcium Indicators

Classic Single-Wavelength Calcium Indicators

Fluorescent Ratiometric Calcium Indicators

Ratiometric, or dual-wavelength, ion indicators are a subcategory of fluorescent dyes utilized for their ability to quantitatively measure intracellular ion concentrations. Compared to single-wavelength indicators, dual-wavelength indicators possess unique spectral properties which are triggered in response to binding its target ion. In the case of ratiometric calcium indicators, such dyes undergo a shift in either their optimum absorption or emission wavelength intensities when binding to free Ca²⁺. For example, dual-excitation Ca²⁺ indicators exhibit two peak excitation wavelengths when either bound or free of Ca²⁺. An increase in Ca²⁺ concentration initiates an increase and decrease in the fluorescence emission intensities of the dual-excitation indicator when excited at the Ca²⁺-bound and Ca²⁺-free peak excitation wavelengths, respectively. Using ratios derived from the photometric data gathered, researchers can accurately determine intracellular Ca²⁺ concentrations. Ratioing techniques are advantageous because it reduces effects indicative of uneven dye loading, poor dye retention and photobleaching. Since their introduction in 1985 by Tsien and collaborators, ratiometric indicators have been cited in countless scientific papers and have aided in advances in investigating the role of calcium in cellular regulation (Grynkiewicz et al. 1985).

FLIPR^® Calcium Assays

Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCRs). Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Fluo-8^® AM, Calbryte™ 520, AM, ™ 590, AM and Rhod-4™ AM which can cross cell membrane. Screen Quest™ Calcium Assay Kits provide an optimized assay method for monitoring GPCRs and calcium channels. These assays can be performed in a convenient 96-well or 384-well microtiter-plate format, are readily adaptable to automation and do not require a wash step.

Luminescent Calcium Detection: Coelenterazine and Derivatives

The aequorin complex is comprised of a 22,000-dalton apoaequorin protein, molecular oxygen and the luminophore coelenterazine. When three Ca²⁺ ions bind to this complex, coelenterazine is oxidized to coelenteramide, with a concomitant release of carbon dioxide and blue light. The approximately third-power dependence of aequorin’s bioluminescence on Ca²⁺ concentration allows the measurement of Ca²⁺ concentrations with a broad detection range from ?0.1 µM to >100 µM. Unlike fluorescent Ca²⁺ indicators, Ca²⁺-bound aequorin can be detected without illuminating the sample, thereby eliminating the interference from autofluorescence.

AAT Bioquest offers coelenterazine and several synthetic coelenterazine analogs for reconstituting aequorin in cells that have been transfected with apoaequorin cDNA. In addition to native coelenterazine, we also offer a few derivatives of coelenterazine that confer different Ca²⁺ affinities and spectral properties on the aequorin complex. Recombinant apoaequorin reconstituted with coelenterazine hcp is reported to have the best luminescence overall, with both a high quantum yield and a fast response time. However, intracellular reconstitution of aequorin from coelenterazine analogs can be relatively slow. Aequorins containing the cp, f or h form of coelenterazine exhibit 10–20 times stronger luminescence than that of apoaequorin reconstituted with native coelenterazine. Coelenterazine h has been predominantly used in HTS screening assay for GPCRs.

Zinc Ion Detection

Zinc is the second most abundant transition metal in living organisms after iron. While most Zn²⁺ in the brain is tightly bound, such that free Zn²⁺ levels extracellularly and intracellularly are likely to be picomolar, a subset of glutamatergic neurons possess weakly bound zinc in presynaptic boutons which is released at micromolar levels in response to a variety of stimuli. The intracellular concentration of free Zn²⁺ is extremely low in most cells (<1 nM), with the remainder being bound to proteins or nucleic acids.

Mounting evidence indicates that zinc has multiple roles in cell biology, as a part of metalloenzyme catalytic sites, as a structural component of gene regulatory proteins, and as a free signal ion, particularly in the cortex of the brain. It is of particular importance in the regulation of gene expression, as Zn²⁺ binding proteins account for nearly 50% of the transcription regulatory proteins in the human genome. Zn²⁺ is also functionally active in pancreatic insulin secretion and is a contributory factor in neurological disorders including epilepsy and Alzheimer’s disease. Free Zn2+ is released from metalloprotein complexes during oxidative stress.

The key to the further progress in understanding the multiple roles of zinc is the availability of fluorescent Zn²⁺ indicator systems that permits quantitative determination and imaging of zinc fluxes and levels over a broad concentration range both intracellularly and extracellularly using fluorescence microscopy and flow cytometry. AAT Bioquest offers the classic TSQ, zinquin, as well as our newly developed sensitive Metal Fluor™ Zn 520 to help elucidate the role of Zn²⁺ release and the localization of free or chelatable Zn²⁺ in cells.

Zinc Indicators Selection Guide