Human MR Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB00501	—
3 x-32 assays in-96 well format	IB00501-32	—
1 x-384 well format assays	IB00502	—

SIZE	SKU
1 x-96 well format assays	IB00501
3 x-32 assays in-96 well format	IB00501-32
1 x-384 well format assays	IB00502

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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 Mineralocorticoid Receptor (MR). INDIGO’s MR reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the MR. In addition to MR 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 MR. 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

Ready to Use Upon Receipt
Includes All Needed Components
Contains Transfected Reporter Cells
Eliminates Cell Licensing Fees
Clear, Reproducible Results
Consistent Results Lot to Lot

Product Specifications

Target Type		Nuclear Hormone Receptor
Species		Human
Receptor Form		Native
Assay Mode		Agonist, Antagonist
Kit Components		MR Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) Aldosterone, (ref. agonist; in DMSO) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Orthologs Available		Yes
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

Agonist dose-response analyses of the Human MR. Analyses of MR Reporter Cells using Aldosterone (provided), Cortisol and Dexamethasone (Cayman Chemical). Concentrated stocks of agonists were prepared in DMSO, then serially diluted using CSM. Final assay concentrations for each agonist were: 4000, 1000, 250, 62.5, 15.6, 3.91, 0.977 and 0 pM. Luminescence was quantified and average relative light units (RLU) and corresponding standard deviation (SD), % coefficient of variation (%CV), and Relative Percent Activation were determined for each treatment concentration (n = 4). Z’ values were calculated as described by Zhang, et al. (1999). GraphPad Prism software was used to perform the least squares method of non-linear regression and to determine EC50 values. The low %CV and high Z' score confirm the robust performance of this MR Assay and its suitability for HTS Applications.

Human MR antagonist assay performance was validated using the reference antagonists Canrenone (Cayman Chemical), Spironolactone, RU26752, RU28318 and Eplerenone (all from Tocris). Assay setup and quantification of MR activity were performed following the protocol described in this Technical Manual. Human MR Reporter cells were co-treated with a fixed ~EC80 concentration of Aldosterone (140 pM) and varying concentrations of the respective antagonist compounds. 'No antagonist' controls were included. Assay plates were incubated for 22 hours, then processed to quantify MR activity for each treatment condition.

Target Background

The mineralocorticoid receptor (MR), also called aldosterone receptor, is a receptor with high affinity for mineralocorticoids. It belongs to the steroid hormone receptor family where the ligand diffuses into cells, interacts with the receptor, and results in a signal transduction affecting specific gene expression in the nucleus. The gene for the NR3C2 (located on chromosome 4q31.1-31.2) encodes for the 107 kDa MR protein.

MR is expressed in many tissues, such as the kidney, colon, heart, central nervous system (hippocampus), brown adipose tissue, and sweat glands. In epithelial tissues, its activation leads to the expression of proteins regulating ionic and water transports (mainly the epithelial sodium channel or ENaC, Na+/K+ pump, serum and glucocorticoid induced kinase or SGK1) resulting in the reabsorption of sodium, and as a consequence an increase in extracellular volume, increase in blood pressure, and an excretion of potassium to maintain a normal salt concentration in the body.

The receptor is activated by mineralocorticoids such as aldosterone and deoxycorticosterone as well as glucocorticoids, like cortisol and cortison. It also responds to some progestins. Spironolactone and eplerenone are mineralocorticoid receptor antagonists. Activation of the mineralocorticoid receptor, upon the binding of its ligand aldosterone, results in its translocation to the cell nucleus, homodimerization and binding to hormone response elements present in the promoter of some genes. This results in the complex recruitment of the transcriptional machinery and the transcription into mRNA of the DNA sequence of the activated genes.

INDIGO’s Mineralocorticoid Receptor Reporter Assay Systems utilize proprietary mammalian cells engineered to express human NR3C2 protein, commonly referred to as MR.

The principle application of this assay product is in the screening of test samples to quantify functional activities, either agonist or antagonist, that they may exert against the human mineralocorticoid receptor.