Human TGFbR Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB12001	—
3 x-32 assays in-96 well format	IB12001-32	—
1 x-384 well format assays	IB12002	—

SIZE	SKU
1 x-96 well format assays	IB12001
3 x-32 assays in-96 well format	IB12001-32
1 x-384 well format assays	IB12002

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Specifications
Data
Target Background
Documentation
Overview
Citation
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Transforming Growth Factor beta Receptors I & II (TGFbR). INDIGO’s TGFbR reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of TGFbR. In addition to TGFbR 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 TGFbR. 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		TGFbR Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) TGF-b1, (ref. agonist; PBS/0.1% BSA ) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Orthologs Available		No
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

Agonist dose-response of the TGFβR. Dose-response analyses of TGFβR Reporter Cells were performed according to the protocol provided in this Technical Manual. TGFβR Reporter Cells were treated with TGF-β using a range of 7 concentrations (n = 4/conc.) generated in 3-fold decrements: 3.0, 1.0, .33, .11, .037, .012 and 0.0041 ng/mL, and including ‘untreated’ (i.e., ‘vehicle only’) control wells, as described in Appendix 1 of the assay technical manual. Luminescence/well was quantified and the average relative light units (RLU) and corresponding standard deviation (SD), percent coefficient of variation (%CV) and Fold-Activation values were determined for each treatment concentration. Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression analyses and IC50 calculations were performed using GraphPad Prism software. These data confirm the robust performance of this TGFβR Assay and demonstrate its suitability for use in HTS applications.

200 µl / well of TGFβR Reporter Cell suspension was dispensed into the 96-well assay plate, which was then placed in a cell culture incubator for four hours. Approximately 30 minutes before the end of the 4 hr pre-culture period, treatment media were prepared by first supplementing CSM with TGF-β1 to a final concentration of 0.6 ng / ml (an approximate EC80 concentration), then using that medium to prepare serial dilutions of the reference antagonists A83-01, SD208, LY36497 and SB431542 (all from Tocris). For each drug, seven treatment concentrations were prepared in 4-fold decrements: 2000, 500, 125, 31.3, 7.81, 1.95 and 0.488 nM. At the end of the pre-culture period media were discarded from the assay wells and 200 µl /well of respective treatment media were dispensed (n = 4/conc.), including ‘vehicle only’ control wells. Residual DMSO was ≤ 0.1% per well in the assay plate. Following a 22 hr incubation period media were discarded, Luciferase Detection Reagent was added, and Luminescence per well was quantified. Values of average relative light units (RLU) and corresponding standard deviation (SD), percent coefficient of variation (%CV), Fold-Inhibition, and % Inhibition were determined for each treatment concentration. Depicted is a plot of % Inhibition of TGFβR vs. Log10[nM] concentration of the various drugs. Non-linear regression analyses and IC50 calculations were performed using GraphPad Prism software.

Target Background

The TGFβ assay uses proprietary human cells that provide constitutive expression of the Human type I and type II Transforming Growth Factor beta Receptors (TGFβ I/II), both of which are transmembrane serine/threonine kinase receptors. Receptor activation results when TGFβ binds to RII, which then phosphorylates and forms a heterodimer complex with RI. The mechanisms of TGFβ-RI/RII activation and ensuing signal transduction cascade have been well studied and involve the phosphorylation and interplay of a variety of Smad regulatory proteins and transcription factors. Additionally, cross-talk between the activated TGFβ and NF-kB signal transduction pathways have been characterized.

INDIGO’s Reporter Cells include the luciferase reporter gene functionally linked to tandem TGFβ Response Sequences (TRS) derived from the human plasminogen activator inhibitor-1 (PAI-1) promoter, a well-characterized TGFβ responsive target gene. The TRS sequences are readily bound by activated dimeric Smad3/Smad4 to initiate formation of a transcription complex. This binding leads to the activation of downstream substrates and regulatory proteins, including transcription of different target genes that function in differentiation, chemotaxis, proliferation, and activation of many immune cells. Among TGFβR’s functions is the regulation of inflammatory processes, particularly in the gut. It also plays a crucial role in stem cell differentiation, as well as T-cell regulation and differentiation.

Quantifying changes in luciferase activity in drug-treated cells versus untreated (or vehicle-treated) reporter cells provides a sensitive surrogate measure of drug-induced changes in TGFβR activity. The principal application of this TGFβ receptor assay is in the screening of compounds / drug candidates to quantify any functional activity, either agonist or antagonist, that they may exert against the human TGFβ receptor.