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FLUOFORTE® Calcium assay kit

A Brighter, More Robust Fluorescent Assay for Calcium Mobilization
ENZ-51017 10x96 tests 347.00 USD
Do you need bulk/larger quantities?
For larger 100 plates size, see ENZ-51016.
  • 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
FLUOFORTE® 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.
ENZ-51016-17 Fig1 webimage 02
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 01
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.
MOA ENZ-51016-17 webimage 01
FLUOFORTE® dye increases in fluorescent intensity when bound to calcium.
ENZ 51017 spectra
Excitation (solid line) and emission (dotted line) spectra of FLUOFORTE® dye
Please mouse over
ENZ-51016-17 Fig1 webimage 02 ENZ-51016-17 Fig2 webimage 01 MOA ENZ-51016-17 webimage 01 ENZ 51017 spectra

Product Specification

Applications:Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS
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 10 x 96-well plates
Handling:Protect from light. Avoid freeze/thaw cycles.
Shipping:Shipped on Dry Ice
Short Term Storage:-20°C
Long Term Storage:-20°C
Contents: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
Technical Info/Product Notes: 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

12(S)-HETE increases intracellular Ca2+ in lymph-endothelial cells disrupting their barrier function in vitro; stabilization by clinical drugs impairing calcium supply: C.H Nguyen, et al.; Cancer Lett. 380, 174 (2016), Application(s): Intracellular Calcium assay, Abstract;
NecroX‐5 suppresses IgE/Ag‐stimulated anaphylaxis and mast cell activation by regulating the SHP‐1‐Syk signaling module: X. Li, et al.; Allergy. 71, 198 (2016), Abstract;
WIF1 re-expression in glioblastoma inhibits migration through attenuation of non-canonical WNT signaling by downregulating the lncRNA MALAT1: I. Vassallo, et al.; Oncogene 35, 12 (2016), Application(s): Assay for microplates, Abstract;
15,16-Dihydrotanshinone I suppresses IgE-Ag stimulated mouse bone marrow-derived mast cell activation by inhibiting Syk kinase: X. Li, et al.; J. Ethnopharmacol. 169, 138 (2015), Application(s): Fluorometric Imaging, Abstract;
ASM-024, a piperazinium compound, promotes the in vitro relaxation of β2-adrenoreceptor desensitized tracheas: E. Israël-Assayag, et al.; PLoS One. 10, e0120095 (2015), Application(s): Microplate reader using human bronchial smooth muscle cells (HBSMC), Abstract; Full Text
Fluorescence/luminescence-based markers for the assessment of Schistosoma mansoni schistosomula drug assays: G. Panic, et al.; Parasit. Vectors. 8, 624 (2015), Application(s): Microplate reader using newly transformed Schistosomula, Abstract; Full Text
Neuroprotective and neuroregenerative effects of nimodipine in a model system of neuronal differentiation and neurite outgrowth: K. Bork, et al.; Molecules 20, 1003 (2015), Application(s): Analysis of rat adrenal PC12 cells, Abstract; Full Text
NF-κB contributes to MMP1 expression in breast cancer spheroids causing paracrine PAR1 activation and disintegrations in the lymph endothelial barrier in vitro: C.H. Nguyen, et al.; Oncotarget. 6, 39262 (2015), Application(s): Microplate reader using lymph endothelial cells (LEC), Abstract; Full Text
Supplemental Online Information: Dual Optical Recordings for Action Potentials and Calcium Handling in Induced Pluripotent Stem Cell Models of Cardiac Arrhythmias Using Genetically Encoded Fluorescent Indicators: L. Song, et al.; Stem Cells Transl. Med. 4, 468 (2015), Application(s): Confocal Microscopy, Abstract;
Low anticoagulant heparin blocks thrombin-induced endothelial permeability in a PAR-dependent manner: J.N. Gonzales, et al.; Vascul. Pharmacol. 62, 63 (2014), Application(s): Microplate reader using human pulmonary artery endothelial cells (HPAEC), Abstract; Full Text
Protein kinase CK2 inhibition induces cell death via early impact on mitochondrial function: F. Qaiser, et al.; J. Cell. Biochem. 115, 2103 (2014), Application(s): Microplate reader using PC3-LN4 cells, Abstract; Full Text
Adipokinetic hormone exerts its anti-oxidative effects using a conserved signal-transduction mechanism involving both PKC and cAMP by mobilizing extra- and intracellular Ca2+ stores: A. Bednarova, et al.; Comp. Biochem. Physiol. C Toxicol. Pharmacol. 158, 142 (2013), Abstract;
Characterization of a novel proinflammatory effect mediated by BK and the kinin B 2 receptor in human preadipocytes: R.M. Catalioto, et al.; Biochem. Pharmacol. 86, 508 (2013), Abstract;
Identification and functional studies of a new Nrf2 partner IQGAP1: a critical role in the stability and transactivation of Nrf2: J.H. Kim, et al.; Antioxid. Redox Signal. 19, 89 (2013), Application(s): Fluorescence microscopy on HeLa cells, Abstract; Full Text
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;
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
Regulatory T cells from IL-10-deficient mice fail to suppress contact hypersensitivity reactions due to lack of adenosine production: S. Ring, et al.; J. Invest. Dermatol. 131, 1494 (2011), Abstract;
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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