Human NF-κB Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB09001	—
3 x-32 assays in-96 well format	IB09001-32	—
1 x-384 well format assays	IB09002	—

SIZE	SKU
1 x-96 well format assays	IB09001
3 x-32 assays in-96 well format	IB09001-32
1 x-384 well format assays	IB09002

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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 Nuclear Factor kappa-light-chain enhancer of activated B cells (NF-kB). INDIGO’s NF-kB reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of NF-kB. In addition to NF-kB 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 NF-kB. 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		Transcription Factor
Species		Human
Receptor Form		Native
Assay Mode		Agonist, Antagonist
Kit Components		NFkB Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) PMA, (ref. agonist; in DMSO) 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

PMA and TNFa activation of Human NF-kB. Activation of NF-kB is demonstrated by treating reporter cells with (a.) Phorbol 12-myristate 13-acetate (PMA; provided), and (b.) TNFa (Tocris) for 22 hours, following the protocol for activation assays depicted in Figure 1. Average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n ≥ 4). Fold-activation and Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression and EC50 analyses were performed using GraphPad Prism software. High Z' scores confirm the robust performance of this assay, and its suitability for HTS

Human NF-κB Reporter Cells were treated with ~EC80 of PMA and challenged with the inhibitors Chromomycin A3, Bortezomib, (S)-MG132, PS-1145 and BMS 345541 (all from Cayman Chemical). Reporter cells were treated for 6 hours, following the protocol for inhibition assays depicted in Figure 1.

Target Background

NF-kB is a signal transduction dependent transcription factor. INDIGO’s Human NF-kB Reporter Assay System utilizes proprietary human cells that express NF-kB (nuclear factor kappa-light-chain enhancer of activated B Cells), and contain the luciferase reporter gene functionally linked to upstream NF-kB genetic response elements. Thus, quantifying changes in luciferase expression provides a sensitive surrogate measure of changes in the level of NF-kB activation. This NF-kB reporter cell line is validated to provide a robust dose-dependent activation response when treated with TNFa, or the Protein Kinase C activator Phorbol 12-myristate 13-acetate (PMA). As such, the principle application of this assay system is in the screening of test samples to quantify any functional activities that they may exert to modulate, either induce or suppress, NF-kB activities.