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Human PDGFR a/b Reporter Assay Kit

SIZES SKU
1 x 96-well format assays IB23001
3 x 32 assays in 96-well format IB23001-32
1 x 384-well format assays IB23002

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Platelet-Derived Growth Factor Receptor α and β (PDGFRα/β). INDIGO’s PDGFRα/β reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the Human Platelet-Derived Growth Factor Receptor α and β. In addition to PDGFRα/β 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 PDGFRα/β. 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
  • PDGFRa/b Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • PDGF-BB, (ref. agonist; 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

PDGFRα/β Activation dose response analyses. Activation dose-response assays were performed according to the protocol provided in this Technical Manual. 200 ul / well of PDGFRα/β Reporter Cell suspension was dispensed into the 96-well assay plate, which was then incubated for 4 hours. The concentrated stock of PDGF-BB (provided), PDGF-AB and PDGF-CC (both from Peprotech) were further diluted using CSM to produce treatment media at the desired assay concentrations. The pre-culture media were discarded from the assay wells and 200 ul / 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’6 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.
PDGFRα/β reporter cells were co-treated with an EC80 concentration of the reference activator PDGF-BB and varying concentrations of the PDGFR inhibitors BIBF-1120, Pazopanib, SU 16f, Sorafenib, AG-1296 and SU6668 (all compounds obtained from Cayman Chemical, Ann Arbor MI, USA). The range of determined IC50 values is shown; (n = 3 / conc.). INDIGO’s Live Cell Multiplex (LCM) Assay confirmed that no treatment concentrations were cytotoxic (data not shown). Non-linear regression analyses of RLU vs. Log10[Inhibitor, nM] were plotted and IC50 determinations made using GraphPad Prism software.

Target Background

Platelet-derived growth factor (PDGF), the physiological activator of PDGFRs, consists of four polypeptide members: A, B, C and D. The biologically active forms of PDGF proteins are both homo-dimers and hetero-dimers of disulfide-linked polypeptides. These function to promote cell migration, proliferation, and survival. Binding of dimeric PDGF triggers conformational changes that drive the assembly of homo-dimeric (Rα:Rα, Rβ:Rβ) and/or hetero-dimeric (Rα:Rβ) receptors, and the activation of their respective cytosolic tyrosine kinase domains. Because these reporter cells constitutively express both PDGFRα and PDGFRβ, it is anticipated that all three forms of the activated receptor dimers are present

The tyrosine kinase activities of activated, dimeric PDGFR’s initiate intracellular signaling cascades that include RAS-MAPK, PI3-AKT, PLCγ and STAT pathways2, 3. For example, activation of the PLCγ pathway leads to an increase of intracellular calcium4 . One prominent outcome of the PDGF/PDGFR > PLCγ pathway is that calcineurin, a calcium dependent phosphatase, dephosphorylates and activates the transcription factor NFAT4. It is PDGFR signal transduction via the Ca+2∙calcineurin / NFAT cascade that is exploited by the reporter cells in this assay

The clinical use of recombinant PDGF-BB has led to the successful treatment of chronic or diabetes-related non-healing lower extremity wounds. In addition, PDGF-BB has also been used in clinics for reducing Parkinsonian symptoms. However, dysfunctional PDGFR signaling can lead to a range of physiological disorders. For example, enhanced signaling of PDGFRs has been implicated in the pathogenesis of atherosclerosis, pulmonary fibrosis, angiogenesis, and tumorigenesis. Consequently, the PDGF receptors continue to command much interest as targets for drug development and drug safety screening.

INDIGO’s PDGFRα/β Reporter Cells contain the luciferase reporter gene functionally linked to tandem consensus sequences of NFAT response elements upstream of a minimal promoter. Activated NFAT binds to these response elements to initiate the formation of a complete transcription complex that drives Luc gene expression. PDGF activates PDGFRα/β in a dose-dependent manner, thereby triggering the Ca+2∙calcineurin/NFAT signal transduction pathway. Quantifying relative changes in luciferase activity in the treated reporter cells relative to the untreated cells provides a sensitive, dose-dependent surrogate measure of drug-induced changes in PDGFRα/β activity.

The principal application of this reporter assay is in the screening of test materials to quantify any functional interactions, either activating or inhibitory, that they may exert against PDGFRα/β, or the coupled Ca+2∙calcineurin/NFAT signal transduction pathway.

Also available as a service

Platelet-Derived Growth Factor Receptors alpha & beta