FluoForte™ Calcium assay kit for microplates



ENZ-51016	1 Kit	3'450.00 USD

Homogeneous mix-and-read, no-wash calcium mobilization assay
Load dye in live cells at 37°C or even room temperature for easier automation
Broadly applicable to both GPCR and calcium ion channel targets
Significantly brighter fluorescence intensity than competitors’ no-wash kits
Provides comparable EC50 values as obtained using Fluo-3, Fluo-4 and FLIPR® Calcium 4 assay kits
No quencher dye, so no potential interference from compound ligand-receptor interactions
Larger assay window than other dyes, allows measurements of challenging cell lines and receptors

Enzo Life Sciences’ FluoForte™ Calcium Assay kit provides a homogeneous fluorescence-based assay for detecting intracellular calcium mobilization across a broad spectrum of biological targets. Relative to other commercially available dyes, FluoForte™ dye yields the brightest signal and largest assay window. The kit provides a homogeneous mix-and-read, no-wash calcium mobilization assay. The homogenous cell-based assay for calcium offers fewer steps, lower variability and an easier protocol for adherent and non-adherent cell lines.

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Product Specification

Quantity:	For 100 x 96-well plates

Quality Control:	% purity of Reagent A (FluoForte™ dye) by HPLC is ≥95%.

Kit/Set Contains:	Reagent A (lyophilized FluoForte™ dye), 10 vials Reagent B (dye efflux inhibitor), 10 x 10 ml

Application:	Provides a homogeneous fluorescence-based assay for detecting intracellular calcium mobilization across a broad spectrum of biological targets

Shipping:	SHIPPED ON DRY ICE

Short Term Storage:	-20°C

Long Term Storage:	-80°C

Handling:	Avoid freeze/thaw cycles. Protect from light.

Background / Technical Information:	For the Manual please click here. The FluoForte™ Calcium Assay Kit is a member of the CELLestial^® product line, reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications. CELLestial^® reagents and kits are optimal for use in demanding imaging applications, such as confocal microscopy, flow cytometry and HCS, where consistency and reproducibility are required.

FIGURE 1: FluoForte™ for HTS GPCR Activation Assay. 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.

FIGURE 2: FluoForte™ for HTS Cellular Analysis. ATP dose response curves in CHO-M1 cells. CHO cells were seeded overnight at 40,000 cells per 100µl per well in a 96-well black wall/clear bottom microplate. (A) The cells were incubated with 100µl of reagent from Life Technologies’ Fluo-4 Direct kit™, Molecular Devices’ Calcium 4 kit (both based upon manufacturer’s protocol) or ELS FluoForte™ dye (B) The cells were incubated with reagent from ELS FluoForte™ Calcium assay kit, or with Life Technologies’ Fluo-4 NW kit (based upon manufacturer’s protocol). ATP (20µl/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 3: 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 100µl per well in a 96-well black wall/clear bottom costar plate. The cells were incubated with 100µl of reagent from Life Technologies’ Fluo-4 Direct™ kit, Molecular Devices’ Calcium 4 kit (both based on manufacturers’ protocol) or Enzo Life Sciences’ FluoForte™ Assay kit. ATP (20 µl/well) was added by FlexStation to achieve concentrations of 200nM.

FIGURE 4: FluoForte exhibits significantly brighter fluorescence intensity over Fluo-4 AM and Fluo-3 AM. Hela cells were seeded overnight at 40,000 cells per 100µl per well in a 96-well black wall/clear bottom costar plate. The growth medium was removed and the cells were incubated with 100µl of 4µM 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 200µl HHBS, ATP (20µl/well) was added to achieve concentrations of 100nM with dye efflux inhibitor, then immediately imaged with a fluorescence microscope (Zeiss) using FITC channel.

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General Literature References

Loading and localization of Fluo-3 and Fluo-3/AM calcium indicators in sinapis alba root tissue A. Tretyn, et al. Folia Histochem. Cytobiol. 35 41 (1997) Abstract

Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3 C. Perez-Terzic, et al. Cell Calcium 21 275 (1997) Abstract

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41 M. do Ceu Monteiro, et al. Cytometry 35 302 (1999) Abstract

Signalling specificity in GPCR-dependent Ca2+ signalling K. Kiselyov, et al. Cell Signal. 15 243 (2003) Abstract

High throughput assay technologies for ion channel drug discovery W. Zheng, et al. Assay Drug Dev. Technol. 2 543 (2004) Abstract

Novel fluo-4 analogs for fluorescent calcium measurements V.V. Martin, et al. Cell Calcium 36 509 (2004) Abstract