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FGFR1/2 Reporter Assay Kit

SIZES SKU
1 x 96-well format assays IB21001
3 x 32 assays in 96-well format IB21001-32
1 x 384-well format assays IB21002

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Fibroblast Growth Factor Receptor 1 & 2 (FGFR1/2). INDIGO’s FGFR1/2 reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of FGFR1/2. In addition to FGFR1/2 Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human FGFR1/2. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Clear, Reproducible Results

  • All-Inclusive Assay Systems
  • Exceptional Cell Viability Post-Thaw
  • Consistent Results Lot to Lot

Product Specifications

Target TypeGrowth Factor Receptor
SpeciesHuman
Receptor FormNative
Assay ModeAntagonist
Kit Components
  • FGFR1/2 Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • FGF-Acidic, (ref. activator; in PBS/0.1% BSA )
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Orthologs AvailableNo
Shipping RequirementsDry Ice
Storage temperature-80C

Data

FGFR1/2 Activation dose response analyses. Activation dose-response assays were performed according to the protocol provided in the assay Technical Manual. 200 µl / well of FGFR1/2 Reporter Cell suspension was dispensed into the 96-well assay plate, which was then incubated for 4 hours. The concentrated stock of FGF-Acidic (aka FGF1; provided), FGF-Basic (aka FGF2) and FGF-21 (all from Peprotech) were further diluted using CSM+H to produce treatment media at the desired assay concentrations. As expected, the endocrine growth factor FGF-21 shows no activity. The pre-culture media were discarded from the assay wells and 200 µl per well of the prepared treatment media were dispensed (n = 3/conc.), including ‘untreated’ control wells. Following a 22 hr incubation period treatment media were discarded, Luciferase Detection Reagent was added, and luminescence intensity per well was quantified. Values of average relative light units (RLU) and corresponding values of standard deviation (SD), percent coefficient of variation (%CV), Fold-Activation and Z’ were determined for each treatment concentration. Non-linear regression analyses of Fold Activation vs. Log10[ng/mL] and EC50 determinations were performed using GraphPad Prism software.

Target Background

The family of Fibroblast Growth Factors (FGFs) comprise approximately 23 members that are related by core sequence and structure conservation, with the majority of FGFs being secreted signaling proteins. Secreted FGFs are predominantly autocrine and paracrine factors, with only three members evolved to function as endocrine factors. FGFs bind and activate FGF Receptors (FGFRs) which, themselves, are members of the family of high-affinity tyrosine kinase receptors.

Heparin and heparin sulfate proteoglycans (HSPGs) are essential cofactors for paracrine FGF interactions with FGFRs. The association between paracrine FGFs and HSPGs ensures their limited diffusion and enhanced FGFR binding specificity. In contrast, endocrine FGFs have minimal affinity to heparin. Rather, they require association with members of the Klotho family of proteins as cofactors for efficient binding to their cognate receptors.

The FGFs are broad-spectrum mitogens that, through their receptor interactions, regulate a variety of cellular functions including migration, proliferation, differentiation, metabolism, and survival. In particular, FGF/FGFR signaling plays a critical role in regulating metabolism in the kidney, liver, brain, intestine, and adipose tissues. Perhaps not surprisingly, dysfunctional FGFR signaling can lead to a range of physiological disorders. For example, mutation, amplification, and gene fusion may result in abnormal morphogenesis and the progression of several types of cancer. Consequently, the FGF receptors continue to command much interest as targets for drug development and drug safety screening.

INDIGO’s assay utilizes proprietary human cells that have been engineered to provide constitutive expression of the Human Fibroblast Growth Factors 1 and 2 (FGFR1/2). FGFR1 and FGFR2 are both single-pass transmembrane receptors that contain respective extracellular ligand-binding domains, transmembrane domains, and intracellular tyrosine kinase domains.

INDIGO’s Compound Screening Media is supplemented with heparin, thereby enabling the formation of FGF/Heparin complexes that bind with high affinity to FGFR monomers. This binding interaction triggers conformational changes that drive the assembly of homo-dimeric (R1:R1, R2: R2) and/or hetero-dimeric (R1:R2) receptors, and the activation of their respective cytosolic tyrosine kinase domains.

The tyrosine kinase activities of activated FGFR’s initiate intracellular signaling cascades that include RAS-MAPK, PI3-AKT, PLCγ, and STAT pathways. For example, activation of the PLCγ pathway leads to an increase of intracellular calcium. One prominent outcome of the FGF/FGFR>PLCγ pathway is that calcineurin, a calcium-dependent phosphatase, dephosphorylates and activates the transcription factor NFAT. It is FGFR signal transduction via the Ca+2 calcineurin / NFAT cascade that is exploited by the reporter cells in this assay.

Also available as a service

Fibroblast Growth Factor Receptor 1 & 2 (FGFR1/2)