Human 5-HT2A Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB54001	—
3 x-32 assays in-96 well format	IB54001-32	—

SIZE	SKU
3 x-32 assays in-96 well format	IB54001-32
1 x-96 well format assays	IB54001

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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 the Human 5-Hydroxytryptamine Receptor 2A Reporter Assay System (5-HT2A; HTR2A). INDIGO’s 5-HT2A reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the 5-HT2A. In addition to 5-HT2A 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 5-HT2A. 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
Kit Components		5-HT2A Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) Serotonin (in DMSO) Detection Substrate Detection Buffer 96-well, collagen-coated assay plate (white, sterile, cell-culture ready)
Shelf Life		6 months
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

Activation of 5-HT2A. Activation assays were performed using the reference compounds Serotonin (provided), 2,5-Dimethoxy-4-iodoamphetamine (DOI), Lysergic Acid Diethylamide (LSD), and 25C-NBOH. The absence of signal in Serotonin treated ‘Mock’ cells (which contains the NFAT-Luc reporter vector, but do not express 5-HT2A) confirms that the observed ligand-dependent response is specific to 5-HT2A activation. Luminescence was quantified and values of average (n = 3) relative light units (RLU), corresponding standard deviation (SD), Fold-Activation, and Z’ values were calculated. The least-squares method of non-linear regression was used to plot Fold-Activation vs. Log10 [Compound, nM] and EC50 values were determined using GraphPad Prism software. All chemicals were procured from Cayman Chemical, Ann Arbor MI, USA.

Inhibition of 5-HT2A. 5-HT2A reporter cells were co-treated with an EC80 concentration of the reference activator Serotonin and varying concentrations of the 5-HT2A specific inhibitors Pimavanserin and Volinanserin, as well as a broad antagonist Ketanserin. 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) relative light units (RLU), and corresponding standard deviation (SD) values were calculated. The least-squares method of non-linear regression was used to plot RLU vs. Log10 [Compound, nM] IC50 values were determined using GraphPad Prism software. All chemicals were procured from Cayman Chemical, Ann Arbor MI, USA.

Target Background

Serotonin (5-hydroxytryptamine, 5-HT) receptors comprise seven receptor families (5-HT1 to 5-HT7) and 14 receptor subtypes in humans. With the exception of the 5-HT3 receptor, which is a ligand-gated ion channel, serotonin receptors are G-protein-coupled receptors (GPCRs), with the majority belonging to the class A family. Serotonin receptors are widely expressed throughout the central and peripheral nervous systems and are involved in diverse physiological processes, including cognition, mood, sensory perception, nociception, gastrointestinal function, cardiovascular regulation and sleep. Their broad tissue distribution and functional diversity make them important targets in drug discovery and pharmacological research.

Beyond primary drug discovery, serotonin receptors are also important in secondary pharmacology and safety assessment, where unintended interactions may identify potential off-target liabilities associated with central nervous system, cardiovascular, gastrointestinal, and other adverse pharmacological effects.

Among the serotonin receptors, 5-hydroxytryptomine receptor 2A (HTR2A, a.k.a. 5-HT2A) is an important target in CNS pharmacology and drug discovery. 5-HT2A is primarily coupled to Gq/11 signaling pathway, leading to phospholipase C activation, intracellular calcium mobilization, and downstream transcriptional responses2. The receptor is highly expressed in the cerebral cortex and contributes to the regulation of cognition, perception, mood, and other CNS functions. 5-HT2A is targeted by diverse pharmacological agents, including psychedelic compounds that act as receptor agonists and atypical antipsychotics that exhibit antagonist or inverse agonist activity. Accordingly, 5-HT2A functional assays are useful for characterizing both on-target and off-target serotonergic activity and for evaluating compound potency, efficacy, and functional pharmacology.