FluoForte^® Calcium assay kit for microplates

A Brighter, More Robust Fluorescent Assay for Calcium Mobilization



ENZ-51017	1 Kit	300.00 USD

Dye optimized for superior cell-permeability and retention
Economical alternative developed for use with conventional dual-dispensing microplate readers, i.e. BioTek, BMG Labtech, etc.
Provides EC50 values comparable to Fluo-4 and Calcium 4
2-fold brighter fluorescence vs. Fluo-4
Sensitive dye provides larger assay window allowing for detection of even weak signal compound responses
Better able to detect native levels of GPCR expression in cultured cells
Data suitable for comparison with that obtained using alternative dyes

Fluo-Forte^® Calcium Assay Kits detect mobilization of intracellular calcium utilizing a fluorogenic calcium-binding dye optimized for superior cell-permeability and retention. The self-quenching dye undergoes an electronic change upon binding of calcium, resulting in a several order of magnitude greater fluorescence.

Mechanism of Action
The fluorogenic calcium-binding dye is provided to the cells as an acetoxymethyl (AM) ester, which is cell-permeable. Once inside the cells, the dye is hydrolyzed by intracellular esterases, which leads to generation of a cell membrane impermeable negatively charged form.

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Figure 1: FluoForte exhibits significantly brighter fluorescence intensity than Fluo-4 AM and Fluo-3 AM. Evaluation of relative dye fluorescence. U2OS cells were seeded overnight, growth medium was removed, and cells were incubated with 100 µl of FluoForte® AM, Fluo-3 AM, or Fluo-4 AM in HHBS at 37 °C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µl HHBS, and ATP (20 µL/well) was added to achieve concentrations of 200 nM with dye efflux inhibitor. Cells were then immediately imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

Figure 2: Obtain robust signal intensity. ATP dose response curves in CHO-M1 cells, expressing P2Y endogenous receptors. CHO cells were seeded overnight at 40,000 cells per 100 µl per well in a 96-well black wall/clear bottom microplate. The cells were incubated with 100 µl of Life Technologies’ Fluo-4 Direct™ kit, Molecular Devices’ Calcium 4 kit (both based upon manufacturer’s protocol) or FluoForte® dye. ATP (20µl/well) was added by FlexStation to achieve the final indicated concentrations. Comparable ATP EC50 values were obtained using all three dyes, while FluoForte generated the highest intensity signal.

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Product Specification

Quantity:	For 10 x 96-well plates

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

Kit/Set Contains:	Reagent A (lyophilized FluoForte^® dye), 1 vial Reagent B (dye efflux inhibitor), 10 x 1 ml Reagent C (Hanks’ buffer with 20mm HEPES), 100 ml

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

Short Term Storage:	-20°C

Long Term Storage:	-80°C

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

Background / Technical Information:	Measurement of transient calcium mobilization from intracellular stores in response to the activation of G protein-coupled receptors (GPCRs) is considered a standard approach to the pharmacological characterization of receptors and compounds, frequently implemented in primary screening and lead development programs. 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.

General Literature References

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;

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

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;