Calcium Assays

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Calcium Assays

Find ABD Bioquest reagents for assays of intracellular calcium at good prices here at www.biomol.de:

Ca2+ Detection by Green Fluorescence

Quest™ Fluo-8 Calcium Assay Kit for Cellular Analysis: Ideal as calcium assay, brighter than Fluo-3 and Fluo-4.

Ca2+ Detection by Red Fluorescence

Rhod-4: The Brightest and Most Sensitive Red Fluorescent Ca2+ Indicator: Large assay window - 10 times better than Rhod-2 AM for HTS applications.

New: Robust Homogeneous Fluorescence-based Calcium Detection with Cal-520™

Cal-520™ enables probenecid-free Ca2+ assays with significantly higher S/N ratio than any other commercially available fluorescent Ca2+ assays.

Comparison of Fluo-3 AM, Fluo-4 AM and Cal-520 AM

Cal-520™ AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520™ AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal 520™ AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520™. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520™ AM an ideal indicator for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520(TM) calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.

Cal 520™ AM technology has the following benefits and features:

Enable probenecid-free Ca2+ assays.
Significantly higher S/N ratio than any other commercially available fluorescent Ca2+ assays.
Essentially identical spectra to Fluo-3, Fluo-4, and Fluo-8™.

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Quest™ Fluo-8 Calcium Assay Kit for Cellular Analysis

Ideal as calcium assay, brighter than Fluo-3 and Fluo-4.

Utilizing the brightest and most sensitive fluorescent Ca2+ indicator, Screen Quest™ Fluo-8 Calcium Assay Kit provides much higher signal intensity and very large fluorescence intensity increase in response to Ca2+ binding. AAT Bioquest’s homogeneous fluorescence-based assay kit detects calcium mobilization across a broad spectrum of biological targets. Easy-to-use protocol does not require a wash step or exogenous addition of a quencher dye, which could adversely affect receptor-ligand interaction kinetics.

Other widely used calcium ion indicators, such as Fluo-3, Fluo-4 and Calcium 4 dyes, have relatively weak fluorescence signals limiting their application in high-throughput screening applications. Quest Fluo-8™ dye offers superior measurement of challenging cell lines and receptors.

Features	Benefits
Load dye in live cells at room temperature (or 37°C)	Room temperature is more compatible with HTS applications
Homogeneous mix-and-read, no-wash calcium mobilization assay	Washing cells introduces errors
Significantly brighter fluorescence intensity	2X brighter than Fluo-4, Fluo-4 Direct, Fluo-4 NW, FLIPR® Calcium 4 assay kits 4X brighter than Fluo-3
EC₅₀ values (half maximal effective concentration)	Comparable with Fluo-3, Fluo-4, Fluo-4 Direct™ and FLIPR® Calcium assay kits
No quencher dye	No potential interference from compound ligand-receptor interactions
Large assay window compared to other dyes	Easily detects low calcium mobilization in challenging cell lines and receptors
Maximum excitation at ~490 nm	Readily adopted into established protocols and instrument workflows
Rigorous Quality Control	Controls and eliminates non-specific assay artifacts

Summary

Compared to other commercial calcium assay kits based on either Fluo-3 or Fluo-4, AAT Bioquest\'s Screen Quest™ Calcium Assay Kits have the following advantages for HTS applications:

Broad Applications: work with both GPCR and calcium channel targets.
Convenient Spectral Wavelengths: maximum excitation @ ~490 nm; maximum emission @ ~514 nm.
Faster Dye Loading: dye loading at room temperature (rather than 37 ºC required for Fluo-4 AM).
No Wash Required and No Quencher Interference with Your Targets.
Robust Performance: enable calcium assays that are impossible with Fluo-4 AM or Fluo-3 AM.
Strongest Signal Intensity: 2 times brighter than Fluo-4 AM; 4 times brighter than Fluo-3 AM.

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Protocols:

Rhod-4: The Brightest and Most Sensitive Red Fluorescent Ca2+ Indicator

Since the 1920s, scientists have attempted to measure Ca2+, but few were successful due to limited availability of Ca2+ probes. The first reliable measurements of Ca2+ were 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 the rhodamine-based Ca2+ reagents (such as Rhod-2) have been the most valuable longer wavelength dye for measuring Ca2+ with red fluorescence.

Although Rhod-2 has been the most popular red fluorescent Ca2+ indicator, its mitochondrial localization and high basal Ca2+ signal in cells have severely limited its cellular applications. In addition, the less optimal excitation of Rhod-2 at 488 nm makes it less robust to use with some instruments (such as FLIPR™) that have only 488 nm excitation light source. Quest Rhod-4™ serial calcium detection reagents have been developed to address these limitations of Rhod-2. The absorption and emission peaks of Quest Rhod-4™ reagents are 530 nm and 555 nm, respectively. Although Rhod-4 has maximum absorption at 530 nm, its absorption at 488 nm is quite strong. It is quite unique that Quest Rhod-4™ can be well excited with an argon ion laser at 488 nm besides the longer wavelength 514 nm, 532 nm and 546 nm excitations. Quest Rhod-4™ emits fluorescence (at wavelengths 555 nm) that increases with increasing Ca2+. Quest Rhod-4™ is determined to undergo a >200-fold increase in fluorescence upon binding Ca2+. Because the range of increase in Ca2+ in many cells after stimulation is generally 5- to10-fold, Quest Rhod-4™ is an excellent probe to use with high sensitivity in this region. The Kd of Quest Rhod-4™ is estimated to be 525 nM (22°C, pH 7.0 –7.5), but this value may be significantly influenced by pH, viscosity, and binding proteins in vivo conditions.

Longer Wavelengths: excitations @ 488, 514, 532 & 546 nm; emission @ ~555 nm.
No Wash Required and No Quencher Interference with Your Targets.
Robust Performance: enable calcium assays that are impossible with Rhod-2 AM.
Strongest Signal Intensity: 4 times brighter than Rhod-2 AM in fluorescence imaging applications.
Larger Assay Window: 10 times better than Rhod-2 AM for HTS applications.

Cat #	Product Name	Unit Size
ABD-21121	Quest Rhod-4™, AM	5x50 µg
ABD-21123	Quest Rhod-4™, AM	20x50 µg
ABD-21128	Quest Rhod-4™, sodium salt	5x50 µg
ABD-36331	Screen Quest™ Rhod-4 NW Calcium Assay Kit Medium Removal	10 Plates
ABD-36334	Screen Quest™ Rhod-4 NW Calcium Assay Kit 1% FBS Growth Medium	10 Plates