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FluoForte® Calcium assay kit for microplates

A Brighter, More Robust Fluorescent Assay for Calcium Mobilization
ENZ-51016 1 Kit 2,245.00 USD
For smaller size, see ENZ-51017.
  • Dye optimized for superior cell-permeability and retention
  • Economical alternative developed for use with conventional dual-dispensing microplate readers, i.e. BioTek, BMG Labtech, etcetera.
  • 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. In the absence of calcium, the calcium-binding moiety portion of the probe quenches the fluorescence of the fluorophore portion of the probe by photo-induced electron transfer. Binding of calcium relieves quenching and results in a several order of magnitude increase in the fluorescence emission intensity, with no shift in wavelength. The dye is capable of binding to physiologically relevant levels of calcium and increases in intracellular calcium lead to an increased fluorescence signal, which is readily measurable.
MOA ENZ-51016-17 webimage
ENZ-51016-17 Fig1 webimage 01
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.
ENZ-51016-17 Fig2 webimage
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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MOA ENZ-51016-17 webimage ENZ-51016-17 Fig1 webimage 01 ENZ-51016-17 Fig2 webimage

Product Specification

Application Notes:Provides a homogeneous fluorescence-based assay for detecting intracellular calcium mobilization across a broad spectrum of biological targets
Quality Control:% purity of Reagent A (FluoForte® dye) by HPLC is ≥95%.
Quantity:For 100 x 96-well plates
Handling:Protect from light. Avoid freeze/thaw cycles.
Short Term Storage:-20°C
Long Term Storage:-80°C
Kit/Set Contains:Reagent A (lyophilized FluoForte® dye), 10 vials
Reagent B (dye efflux inhibitor), 10 x 10 ml
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.


Product Literature References

Inula japonica extract inhibits mast cell-mediated allergic reaction and mast cell activation: Y. Lu, et al.; J. Ethnopharmacol. 143, 151 (2012), Application(s): Calcium levels in mouse bone marrow-derived mast cells, Abstract;
Leukotriene C4 induces migration of human monocyte-derived dendritic cells without loss of immunostimulatory function: J. Dannull, et al.; Blood 119, 3113 (2012), Application(s): Intracellular calcium release in human monocyte derived dendritic cells with Tecan Infinite plate reader, Abstract; Full Text
Natural vanadium-containing Jeju groundwater inhibits immunoglobulin E-mediated anaphylactic reaction and suppresses eicosanoid generation and degranulation in bone marrow derived-mast cells: X. Li, et al.; Biol. Pharm. Bull. 35, 216 (2012), Application(s): Calcium levels in mouse bone marrow-derived mast cells, Abstract; Full Text
Saucerneol D inhibits eicosanoid generation and degranulation through suppression of Syk kinase in mast cells: Y. Lu, et al.; Food Chem. Toxicol. 50, 4382 (2012), Application(s): Calcium levels in mouse bone marrow-derived mast cells, Abstract;
Amplification of cancer cell apoptosis in photodynamic therapy-treated tumors by adjuvant ceramide analog LCL29: M. Korbelik, et al.; Lasers Surg. Med. 43, 614 (2011), Application(s): Calcium levels in mouse squamous cell carcinoma, Abstract;
FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ: S. Kumar, et al.; Chemphyschem. 12, 609 (2011), Application(s): Use with HEK293 cells, Abstract; Full Text
The P2Y(2) nucleotide receptor mediates tissue factor expression in human coronary artery endothelial cells: L. Ding, et al.; J. Biol. Chem. 286, 27027 (2011), Application(s): Calcium levels in human coronary artery endothelial cells, Abstract; Full Text

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;

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