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FLUOFORTE® Calcium Assay Kit

Utilizing the brightest and most sensitive fluorescent Ca2+ indicator, FLUOFORTE® Calcium Assay kit provides much higher signal intensity and the largest assay window on the market. Enzo Life Sciences' 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. The assay can be performed in a convenient 96-well or 384-well microplate format and easily adapts to automation.

FLUOFORTE® dye offers superior measurement of challenging cell lines and receptors; unlike other widely used calcium ion indicators in high-throughput screening applications; Fluo-3, Fluo-4 and Calcium 4 dyes have relatively weak fluorescence signals limiting their application in these instances.

 

FLUOFORTE® Calcium Assay is Visibly Brighter

FeaturesBenefits
Load dye in live cells at 37°C or RT 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
Larger assay window than other dyes Easily detects low calcium mobilization in challenging cell lines and receptors
Maximum excitation @ ~490 nmReadily adopted into established protocols and instrument workflows
Stringently manufactured Controls and eliminates non-specific assay artifacts
Figure 1: Hela cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with 100 µl of 4 uM Fluo-3 AM, Fluo-4 AM or FLUOFORTE® AM in HHBS at 37 °C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µl HHBS, ATP (20 µL/well) was added to achieve concentrations of 100 nM with dye efflux inhibitor, then immediately imaged with a fluorescence microscope (Zeiss) using FITC channel.
 

FLUOFORTE® Calcium assay kit  

A Brighter, More Robust Fluorescent Assay for Calcium Mobilization
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51016 100x96 tests 2,500.00 USD
Do you need bulk/larger quantities?
 

FLUOFORTE® Calcium assay kit  

A Brighter, More Robust Fluorescent Assay for Calcium Mobilization
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51017 10x96 tests 347.00 USD
Do you need bulk/larger quantities?
 

HTS GPCR Activation Assay

GPCR activation can be detected by direct measurement of the receptor mediated cAMP accumulation or changes in intracellular Ca2+ concentration. GPCR targets that couple via Gq produce an increase in intracellular Ca2+ that can then be measured with FluoForte and a fluorescence microplate reader. FluoForte™ has been validated on such instruments as FLIPRTETRA, Flex-Station® II and 3, Perkin Elmer Cellux®, and Hamamatsu FDSS6000/7000 models. Besides its robust applications for GPCR targets, our FluoForte™ Calcium Assay Kit can also be used for characterizing calcium ion channels and screening calcium ion channel-targeted compounds.



FIGURE 1: FLUOFORTE™ enters cell as a membrane permeable acetoxymethyl (AM) ester. Once inside the cell, FLUOFORTE is hydrolyzed by intracellular esterases. Cell membrane impermeable, negatively charged form of FLUOFORTE- is now capable of binding with Ca2+. Some cell lines express an organic anion transporter, which leads to the export of negatively charged FLUOFORTE- . This can be prevented by adding Dye Efflux Inhibitor, a transport inhibitor.

 

Cellular Analysis Application



FIGURE 1: ATP Dose Response Curves in CHO-M1 cells. CHO cells were seeded overnight at 40,000 cells per 100 µl per well in a 96-well black wall/clear bottom microplate. (A) The cells were incubated with 100 µl of reagent from Life Technologies’ Fluo-4 Direct kit™, Molecular Devices’ Calcium 4 kit (both based upon manufacturer’s protocol) or Enzo’s FLUOFORTE™ dye (B) The cells were incubated with reagent from Enzo’s FLUOFORTE™ Calcium Assay kit, or with Life Technologies’ Fluo-4 NW kit (based upon manufacturer’s protocol). ATP (20 µl/well) was added by FlexStation to achieve the final indicated concentrations. No significant difference in EC50 of ATP for FLUOFORTE™ assay kit, Fluo-4 Direct™ and Calcium 4 was observed (shown in A), and also comparable EC50 values were observed between FLUOFORTE™ Assay kit and Fluo-4 NW (shown in B). In all cases FLUOFORTE™ Assay kit generated the highest intensity signal.



FIGURE 2: Comparisons of FLUOFORTE™ Assay Kit, Fluo-4 Direct™, and Calcium 4 detection of intracellular calcium mobilization in CHO-M1 cells. CHO cells were seeded overnight in 40,000 cells per 100 µl per well in a 96-well black wall/clear bottom costar plate. The cells were incubated with 100 µl of reagent from Life Technologies’ Fluo-4 Direct™ kit, Molecular Devices’ Calcium 4 kit (both based on manufactures’ protocol) or Enzo’s FLUOFORTE™ Assay kit. ATP (20 µL/well) was added by FlexStation to achieve concentrations of 200 nM.

 

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