Fibroblast Growth Factor Receptors 1 & 2 for Paracrine FGF Signaling (FGFR1/2)
|Product Family||Product Number||Product Description||Technical Manual|
|IB21001-32||Human FGFR1/2 Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB21001||Human FGFR1/2 Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB21002||Human FGFR1/2 Reporter Assay System, 1 x 384-well format assays||Technical Manual|
Human Fibroblast Growth Factor Receptors 1 and 2 Assay Kit
This Human Fibroblast Growth Factor Receptor 1 and 2 (FGFR1/2) assay kit is an all-inclusive firefly luciferase reporter assay system that includes in addition to FGFR1/2 Reporter Cells, an optimized culture medium for use in reviving the cryopreserved cells, a culture medium supplemented with heparin for use in preparing test sample treatments, the physiological activator FGF-Acdic (aka FGF1), Luciferase Detection Reagents, and a cell culture-ready assay plate.
FGFR 1/2 Reporter Cells are transiently transfected and prepared as frozen stocks using INDIGO's proprietary CryoMite™ process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup.
INDIGO’s FGFR 1/2 assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set.
FGFR 1/2 assay kits are offered in different assay formats to accommodate researchers' needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
Human Fibroblast Growth Factor Receptors 1 and 2 Assay Services
The primary application of INDIGO's cell-based FGFR1/2 assays are to quantitatively assess the bioactivity of a test compound as an agonist or antagonist of the receptor. Service assays include a positive control reference compound and 'vehicle' control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online "Request a Quote" form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
Human Fibroblast Growth Factor Receptors 1 and 2 Assay 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.
Synonyms: Fibroblast Growth Factor Receptor, Fibroblast Growth Factor Receptor 1, Fibroblast Growth Factor Receptor 2, FGFR1, FGFR2, FGFR
Human Fibroblast Growth Factor Receptors 1 and 2 Assay 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.