Human PDGFRβ Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB23201	—
3 x-32 assays in-96 well format	IB23201-32	—

SIZE	SKU
3 x-32 assays in-96 well format	IB23201-32
1 x-96 well format assays	IB23201

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Specifications
Data
Target Background
Documentation
Overview
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Platelet-Derived Growth Factor Receptor β (PDGFRβ). INDIGO’s PDGFRβ reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the Human Platelet-Derived Growth Factor Receptor β. In addition to PDGFRβ 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 PDGFRβ. 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 Type		Growth Factor Receptor
Species		Human
Receptor Form		Native
Assay Mode		Antagonist
Kit Components		PDGFRβ Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) PDGF-BB, 10.0 ug/ml Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

PDGFRβ Activation dose response analyses. Activation dose-response assays were performed according to the protocol provided in this Technical Manual. 200 ul / well of PDGFRβ 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; Peprotech), was 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-hour 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

This assay utilizes proprietary human cells that have been engineered to provide constitutive expression of the Human Platelet-Derived Growth Factor Receptor β, referred to herein as PDGFRβ. PDGFRβ is a single-pass transmembrane receptor that contain respective extracellular ligand-binding domains, transmembrane domains, and intracellular tyrosine kinase domains.

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 (Rβ:Rβ) receptors, and the activation of their respective cytosolic tyrosine kinase domains.

The tyrosine kinase activities of activated, dimeric PDGFR’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 PDGF/PDGFR > PLCγ pathway is that calcineurin, a calcium-dependent phosphatase, dephosphorylates and activates the transcription factor NFAT. It is PDGFR signal transduction via the Ca+2∙calcineurin / NFAT cascade that is exploited by the reporter cells provided in this kit.

INDIGO’s PDGFRβ 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.