Human ADRA2A Reporter Assay Kit

SIZE	SKU	PRICE
1 x-384 well format assays	IB34002	—
1 x-96 well format assays	IB34001	—

SIZE	SKU
1 x-384 well format assays	IB34002
1 x-96 well format assays	IB34001

Specifications
Data
Target Background
Documentation
Overview
Citation
Related Products

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Adrenoceptor Alpha 2A (ADRA2A). INDIGO’s ADRA2A reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the ADRA2A. In addition to ADRA2A 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 ADRA2A. 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		GPCR
Species		Human
Receptor Form		Hybrid
Assay Mode		Agonist, Antagonist
Kit Components		Human ADRA2A Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) Brimonidine, (ref. agonist; in DMSO) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

Activation of ADRA2A. Reporter cells were treated with the reference activators Brimonidine (a.k.a. UK14,304; provided), Guanabenz, Clonidine, Moxonidine, Guanfacine, Epinephrine, Norepinephrine, and Lofexidine. The absence of signal in Brimonidine treated ‘Mock’ cells (which contains the NFAT-Luc reporter vector, but do not express ADRA2A) confirms that the observed ligand-dependent response is specific to ADRA2A activation. Luminescence was quantified and values of average (n = 3) RLU, standard deviation (SD), Fold-Activation or % Inhibition, and Z’ values were calculated. The least-squares method of non-linear regression was used to plot activity changes vs. Log10 [Compound, nM], and EC50 /IC50 values were determined, using GraphPad Prism software.

Inhibition of ADRA2A. Reporter cells were co-treated with an EC80 concentration of the reference activator Brimonidine and varying concentrations of the α2 adrenoceptors selective inhibitors Rauwolscine, Yohimbine, Atipamezole, BRL44408, and the general alpha adrenoceptor inhibitors WB4101, BMY7378, and Prazosin. INDIGO’s Live Cell Multiplex (LCM) Assay confirmed that no treatment concentrations were cytotoxic (data not shown). Luminescence was quantified and values of average (n = 3) RLU, standard deviation (SD), Fold-Activation or % Inhibition, and Z’ values were calculated. The least-squares method of non-linear regression was used to plot activity changes vs. Log10 [Compound, nM], and EC50 /IC50 values were determined, using GraphPad Prism software.

Target Background

The adrenoceptors (a.k.a. adrenergic receptors) mediate the action of the sympathetic nervous system and are activated in response to “fight-or-flight” signals. They are divided into three types, adrenoceptor α1-, α2-, and β. Each type is further composed of three subtypes resulting in 9 different types (α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, and β3). Adrenoceptors belong to the G-Protein-coupled receptor (GPCR) family. They all display the characteristic seven transmembrane helices, the extracellular loops which contribute to ligand binding, and the intracellular carboxy tail that associates with trimeric G proteins. All nine types of adrenoceptors are activated by the same endogenous catecholamines (epinephrine and norepinephrine); however, the specificity of their responses depends on the G-proteins and effector systems they associate with in a tissue and time specific manner.

Adrenoceptor Alpha 2A (ADRA2A) is expressed in the central nervous system and in peripheral organs such as spleen, kidney, heart, aorta, skeletal muscle, lung and liver. ADRA2A agonists are used in general anesthesia and in the treatment of opiate withdrawal, attention deficit and hyperactive disorder (ADHD), post-traumatic stress disorder (PTSD), and Tourette’s syndrome. ADRA2A also plays a role in the regulation of blood pressure, in the regulation of glucose metabolism, and in some types of cancer.