Calcium Indicators

Cell-based calcium flux assays are widely used in high throughput screening (HTS) to determine agonist-stimulated and antagonist-inhibited signaling through G protein-coupled receptors (GPCRs), a large family of integral membrane proteins and an important target class in drug discovery. Accurate measurements of intracellular calcium concentration and mobilization allows for a more comprehensive understanding of calcium regulated signaling pathways, cellular functions and pathological processes.

AAT Bioquest has developed the largest collection of calcium indicators, including Fluo-8^® , Cal-520^®, and Calbryte™ calcium indicators, as well as, calcium flux assays to track calcium mobilization with intense fluorescent signals and in a variety of wavelength options ranging from ultraviolet to infrared.

Quantifying Cytosolic Calcium

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Calcium is a ubiquitous messenger impacting a number of signal transduction pathways including calcium-induced changes in protein confirmation, the regulation of cytosolic and organelle calcium levels, the highly localized nature of calcium-mediated signal transduction and its specific roles in excitability, exocytosis, motility, apoptosis, and transcription. These pathways regulate many key cellular processes including neurotransmission, cell proliferation, and muscle contraction. Under normal conditions with respect to the extracellular fluid, the resting cytoplasmic concentration of calcium is relatively low, at or below 100 nM. Calcium signaling is activated either through the release of calcium ions from intracellular stores, or by an influx of extracellular calcium through plasma membrane ion channels. Stimulated cells can increase cytoplasmic calcium concentration up to 500 to 1000 nM.

Fluorescent AM Ester Derivatives for Live Cell Calcium Imaging

Fluorescent indicators that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. To facilitate detecting intracellular calcium mobilization and promote passive diffusion, acetoxymethyl ester (AM ester) functional groups are attached to the core structure of calcium indicators. This increases the indicator's hydrophobicity allowing it to easily penetrate intact plasma membranes, and enhances indicator sensitivity by minimizing non-specific background fluorescence because AM ester conjugated indicators are essentially non-fluorescent and inactive.

Activity is initiated once the probe enters a live cell and only after intracellular esterases remove the AM ester functional groups. Once activated, probes detect calcium through chelation and respond by generating a fluorescence signal relative to the concentration of calcium. Indicators such as Fluo-8^®, AM (Cat No. 21080), Cal-520^®, AM (Cat No. 21131) and Calbryte™ 520, AM (Cat No. 20653) have been extensively used in calcium imaging, calcium flux assays and cell-based high throughput screening (HTS) assays for drug discovery. Calcium indicators are compatible with use in flow cytometry, fluorescence microscopy, and the fluorescence microplate platform. Besides offering fluorescent and luminescent calcium indicators, we also provide several non-fluorescent calcium signaling molecules and chelators for measuring and manipulating intracellular and extracellular calcium.

Single-Wavelength Calcium Indicators

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Single-wavelength calcium indicators are widely used to monitor calcium mobilization, image the spatial dynamics of calcium signaling, and in HTS cell-based pharmacological screening of agonist-stimulated and antagonist-inhibited signaling through GPCRs. These indicators exhibit significant calcium-dependent changes in fluorescence intensity at saturating calcium levels without shifting either their absorbance or emission wavelengths. We've developed a variety of novel calcium indicators, including the Fluo-8^® series, the Cal-520^® series and the Calbryte™ series, designed to trace calcium mobilization with the brightest fluorescent signals and a range of wavelength and affinity options to satisfy many applications.

Key features of single-wavelength calcium indicators

• Minimal background fluorescence at resting calcium levels
• Improved signal-to-background ratios
• Greatly enhanced cellular retention
• Cell-permeant formats for live cell calcium analysis (compatible with imaging, flow cytometry and microplate assays, such as HTS)
• Cell-impermeant formats for loading by microinjection, patch pipette, or pinocytic loading
• Ultraviolet (UV), violet and visible light-excitable formats
• Low-, medium- and high-affinity formats provides a sensitivity range to satisfy many applications

1. K_d = Calcium dissociation constant.
2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
3. F_Ca/F_Free = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

Affinity and specificity are the most important concerns for a calcium probe, in addition to other factors such as cytotoxicity and cell permeability. Color is a nonzero aspect as well, and must suit experimental design. The spectra of red-shifted calcium probes are well separated from those of FITC, Alexa Fluor® 488 and GFP, making them ideal for multiplexing. Rhod-2 is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Other red probes such as Cal-590™ have been developed to improve cell loading and calcium response while maintaining the spectral profile to remain compatible with the common TRITC/Cy3® filter set.

1. K_d = Calcium dissociation constant.

Ratiometric Calcium Indicators

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In comparison to single-wavelength indicators, ratiometric (or dual-wavelength) indicators possess unique spectral properties which are triggered in response to binding calcium. When binding to calcium, ratiometric indicators undergo a shift in either their optimum absorbance or emission wavelength intensities. For example, dual-excitation calcium indicators Fura-2, AM and Fura-8™, AM exhibit two peak excitation wavelengths when either bound or free of calcium. An increase in calcium concentration initiates an increase and decrease in the fluorescence emission intensities of the dual-excitation indicator when excited at the calcium-bound and calcium-free peak excitation wavelengths, respectively.

Key features of ratiometric calcium indicators

• Ratiometric determination produces accurate measurement of intracellular calcium concentrations
• Minimizes effects of uneven dye loading, dye leakage, photobleaching and varying cell thickness (common in mixed populations)
• Delivers more robust and reproducible results
• Indicators available in dual-excitation, dual-emission or dual-excitation and dual-emission formats
• Available as membrane-permeant AM esters (compatible with imaging, flow cytometry and microplate assays, such as HTS)
• Available as membrane-impermeant salt derivatives for loading by microinjection, patch pipette, or pinocytic loading

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1. K_d = Calcium dissociation constant.

1. K_d = Calcium dissociation constant.

1. K_d = Calcium dissociation constant.

Calbryte™ Calcium Indicators

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The Calbryte™ series is a family of the brightest and most robust calcium indicators developed to monitor intracellular calcium mobilization. It includes three novel calcium indicators, Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630, which differ primarily in their absorbance and emission wavelengths. Calbryte™ probes emit fluorescence signals at significantly higher signal-to-background characteristics, and have greatly enhanced cellular retention. The Calbryte™ series has been optimized for use with fluorescence microscopes, fluorescence microplate readers and flow cytometers. They can also be used in high-throughput screening applications.

Key features of Calbryte™ calcium indicators

• Proven performance, superior signal-to-noise ratio and intracellular retention properties
• Calbryte™ probes are significantly brighter than any other calcium indicators under the same conditions
• Greatly improved signal-to-background ratios
• Enhanced intracellular retention, eliminates the use of probenecid
• Fast and convenient cell loading in 45 to 60 minutes
• Homogenous cytosolic localization
• Red-shifted Calbryte™ 590 and Calbryte™ 630 can be used with GFP or other green-fluorescent dyes for multiplexing
• Available as membrane-permeant AM esters (compatible with imaging, flow cytometry and microplate assays, such as HTS)
• Available as membrane-impermeant salt derivatives for loading by microinjection, patch pipette, or pinocytic loading
• Available as a reactive-dye for conjugation to a carrier

1. K_d = Calcium dissociation constant.
2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
3. F_Ca/F_Free = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

Cal-520^® Calcium Indicators

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The Cal-520^® calcium indicators series is the most comprehensive portfolio of fluorogenic calcium-sensitive dyes. It includes six unique indicators, Cal-500™, Cal-520^®, Cal-590™, Cal-630™, Cal-670™ and Cal-770™, which differ primarily in their absorbance and emission wavelengths. Each of the indicators emit minimal fluorescence at resting levels of calcium, and increases its fluorescence intensity >100-fold at saturating calcium concentrations. The Cal-520^® series of indicators are available as membrane-permeant AM esters, and membrane-impermeant salt derivatives and dextran conjugates.

Key features of Cal-520^® calcium indicators

• Higher signal-to-background ratios than Fluo-3, Fluo-4, Rhod-2 and Rhod-5
• Enhanced intracellular retention
• Convenient and efficient cell loading
• Homogenous cytosolic localization
• Red-shifted Cal-590™, Cal-630™, Cal-670™ and Cal-770™ can be used with GFP or other green-fluorescent dyes for multiplexing
• Cal-770™ may be used for in vivo studies
• Available as membrane-permeant AM esters (compatible with imaging, flow cytometry and microplate assays, such as HTS)
• Available as membrane-impermeant salt derivatives for loading by microinjection, patch pipette, or pinocytic loading
• Available as reactive-dyes for conjugation to a carrier

1. K_d = Calcium dissociation constant.
2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
3. F_Ca/F_Free = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.
4. N/D = not determined.

Fluo 8^® Calcium Indicators

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Fluo-8^® AM addresses one of the major limitations common to fluo-based dyes, namely, the harsh conditions required for cell loading. Traditional fluo-based dyes such as Fluo- 2 AM (Cat No. 20495), Fluo-3 AM (Cat No. 21013 and Fluo-4 AM (Cat No. 20550) need to be loaded and incubated with cells at 37°C for up to an hour before a reading can be taken. This not only subjects cells to harsh temperatures, but also requires users to spend time heating and cooling cell cultures. Additionally, it introduces incubation temperature as a possible confounding variable. Fluo-8^® AM resolves all these problem by providing a robust calcium indicator that can be loaded into cells at room temperature. Fluo-8^® AM is less temperature dependent than other fluo-based dyes, allowing for more consistent results under a wider range of experimental conditions. Furthermore, with an incubation time of as little as 20 minutes, Fluo-8^® AM enables fast turnaround for improved productivity. The best part is that even with structural modifications and improvements, Fluo-8^® AM's spectral properties remains unchanged, maintaining the familiar spectra of Fluo-4 AM (ie. Ex/Em = 494/517nm).

Key features of Fluo 8^® calcium indicators

• Faster cell loading: Fluo 8^® can be readily loaded into cells at room temperature. Fluo-3 AM and Fluo-4 AM require a loading temperature of 37°C
• Brighter fluorescence: Fluo 8^® is 2 times brighter than Fluo-4 AM, and 4 times brighter than Fluo-3 AM
• Convenient: Fluo 8^®'s identical spectra to those of Fluo-4 and Fluo-3 makes upgrading easy

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1. K_d = Calcium dissociation constant.
2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
3. F_Ca/F_Free = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

Fluo-3 and Fluo-4 Calcium Indicators

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Fluo-3 is a calcium indicator first introduced in 1989 for use in flow cytometry and confocal laser-scanning microscopy. A fluorescein-like compound, Fluo-3 is effectively non-fluorescent in the absence of free Ca²⁺. Upon binding to calcium, however, Fluo-3 experiences a sharp increase in emission intensity at 525 nm. As an indicator, Fluo-3 has been essential in the study of cell signaling pathways, myocytes and the activation of neurotransmitters.

Fluo-4 builds upon Fluo-3's core structure, but modifying it for increased fluorescence signal strength. The modification in chemical structure causes a blue-shift in Fluo-4's excitation maximum, from 506nm (of Fluo-3) to 494nm. Practically speaking, this 12 nm shift moves Fluo-4's excitation maximum closer to the wavelength of a standard argon laser (ie. 488nm). This results in better excitation and thus, a fluorescent signal that is significantly brighter and more sensitive to changes in calcium concentration.

1. K_d = Calcium dissociation constant.
2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
3. F_Ca/F_Free = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

Calcium Assays for Screening GPCR and Calcium Channel Targets

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Screen Quest™ calcium flux assays are the preferred method in cell-based high throughput screening (HTS) to determine agonist-stimulated and antagonist-inhibited signaling through G protein coupled receptors (GPCRs). To date approximately one third of drugs available on the market are designed to target GPCRs. The Screen Quest™ Calcium Assay offers a convenient no-wash, homogeneous fluorescence-based assay for monitoring GPCRs and calcium channels, and to detect intracellular calcium mobilization across a broad spectrum of biological targets. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and can be easily adapted for automation. Screen Quest™ Calcium Assays are optimized and validated for the following instruments: FDSS^®, FLIPR^®, ViewLux™, NOVOStar, ArrayScan™, FlexStation^® and IN Cell Analyzer.

Key Features of Screen Quest™ Calcium Assays

• Optimized protocols validated for a broad range of microplate readers
• Superior signal intensity (high S/B ratios) and well-to-well uniformity
• Large assay window for assaying challenging cell lines and receptors
• High performance calcium flux assays for 1536 well-plates

GPCR Cell Lines for Drug Discovery and High Throughput Screening

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Screen Quest™ cell lines are a series of cells that have been successfully used in drug discovery and screening environments for studying G-protein-coupled receptors (GPCR) that do not conventionally couple through intracellular calcium. It has been effectively used with the FLIPR, FDSS Systems in conjunction with non-Gq coupled members of many receptors such as chemokine, serotonin, glutamate, dopamine, opioid, vasopressin and α- and ß- adrenergic receptor families. Over 60% of the known GPCR signal through pathways other than Gq which lead to an increase in intracellular calcium, and as genomics reveals more G-protein-coupled receptor targets this trend continues to increase. Screen Quest™ cell lines are used for investigating GPCR that do not conventionally couple through intracellular calcium.
Screen Quest™ cell lines are based on a series of G-protein chimeras, including the promiscuous G-protein, Gα16. The chimeras consist of the alpha subunit of a Gq-protein complex whose 5 carboxy-terminal amino acids have been replaced with those from one of the other G-proteins (either Gαs, Gαi, Gαo, or Gαz). These amino acids are responsible for the coupling of the receptor to its G-protein. Co-expression of these chimeras with specific non-Gq-coupled receptors which normally act through the cAMP pathway may result in the generation of an intracellular calcium signal upon receptor stimulation.

Ordering Information

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