Cannabinoid Type 1 Receptor (CB1R; CNR1)
|Product Family||Product Number||Product Description||Technical Manual|
|IB19001-32||Human CB1R Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB19001||Human CB1R Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB19002||Human CB1R Reporter Assay System, 1 x 384-well format assays|
Human Cannabinoid Type 1 Receptor Assay Kit
This Human Cannabinoid Type 1 Receptor assay kit (CB1R) is an all-inclusive firefly luciferase reporter assay system that includes in addition to CB1 Receptor Reporter Cells, two optimized media for use in recovering the cryopreserved cells and for diluting test samples, the reference activator synthetic cannabinoid CP-55,940, Luciferase Detection Reagents, and a cell culture-ready assay plate.
CB1 Cannabinoid Receptor Reporter Cells are transiently transfected and prepared as frozen stocks using INDIGO's proprietary CryoMite™ process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup.
INDIGO’s CB1 assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set.
CB1 Cannabinoid Receptor assay kits are offered in different assay formats to accommodate researchers' needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
Human Cannabinoid Type 1 Receptor Assay Services
The primary application of INDIGO's cell-based CB1R assays are to quantitatively assess the bioactivity of a test compound as an agonist or antagonist of the receptor. Service assays include a positive control reference compound and 'vehicle' control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online "Request a Quote" form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
Human Cannabinoid Type 1 Receptor Assay Background
The Cannabinoid Type 1 Receptor is one of the two main cannabinoid receptors identified as part of the endocannabinoid system. The CB1 Receptor is highly expressed in the central nervous system, but also in skeletal muscles, gastrointestinal tract, liver, pancreas, skin, reproductive system, and cardiovascular system. The Cannabinoid Type 1 Cannabinoid Receptor is involved in a wide range of physiological and pathophysiological processes related to neurological disorders, energy metabolism, obesity, diabetes, cardiovascular and reproductive disorders, inflammation, and cancer.
The CB1 Receptor is a member of the super-family of G protein-coupled receptors (GPCR). Its structure includes seven transmembrane helices, with an extracellular amino end and an intracellular carboxy tail associated with trimeric G proteins. Upon ligand binding, the receptor undergoes conformational change that triggers the activation of G proteins via an exchange of GDP to a GTP.
Modes of Cannabinoid Type 1 Cannabinoid Receptor signal transduction are diverse and may involve a variety of signaling molecules and kinases that vary based on the type of ligand and the type of cells in which the receptor is expressed. The first signaling pathway described for CB1 receptors involved activation of a pertussis toxin sensitive G protein (Gαi/o), leading to the inhibition of forskolin-stimulated cyclic AMP, activation of G protein-coupled inwardly rectifying potassium channels (GIRKs), and an inhibition of several calcium channels. This is followed by recruitment of β-arrestin, a cytosolic protein involved in receptor desensitization and internalization. However, β-arrestin activation is also involved in the activation of downstream kinases such as MAPK.
CB1R can also couple with stimulatory G proteins such as Gαs and Gαq/11, resulting in the activation of ERK and PLC pathways and increased levels of intracellular calcium. An outcome of CB1R activation is that calcineurin, a calcium-dependent phosphatase, dephosphorylates and activates the transcription factor NFAT. Signal transduction via the Ca+2-calcineurin / NFAT cascade is exploited by the reporter cells used in this assay.
This CB1 Cannabinoid Reporter assay uses proprietary human cells that provide constitutive expression of the Human Cannabinoid Type 1 Receptor.
INDIGO's CB1 Cannabinoid Receptor Reporter Cells contain an engineered luciferase reporter gene functionally linked to tandem NFAT genetic response elements (GRE) and a minimal promoter. Activated NFAT binds to its corresponding GREs to initiate the formation of a complete transcription complex that drives Luc gene expression. Quantifying changes in luciferase activity in the treated reporter cells relative to the untreated cells provides a sensitive, dose-dependent surrogate measure of drug-induced changes in CB1 Cannabinoid Receptor activity. Accordingly, the principal application of this reporter assay is in the screening of test compounds to quantify any functional activities, either activating or inhibitory, that they may exert against CB1R or its associated calcineurin-NFAT signal transduction pathway.
Synonyms: Cannabinoid Receptor 1, CB1R, CNR1, CB1, CNR, CB-R, CB1A, CANN6, CB1K5
Human Cannabinoid Type 1 Receptor Assay DataINDIGO CB1R reporter assay agonist dose response analyses
Agonist dose-response assays were performed using the reference compounds CP 55,940 (provided), HU-210, JWH 210, 4-fluoro MDMB-BUTINACA, EMB-FUBINACA, MAM2201, Δ9-THC, JWH 018, AM1235, WIN 55,212-2 and XLR11 (all compounds from Cayman Chemical, Ann Arbor MI, USA). Luminescence was quantified and values of average relative light units (RLU), corresponding standard deviation (SD), Fold-Activation, and Z’ values were calculated. Non-linear regression analyses of Fold-Activation vs. Log10 [Agonist, nM] were plotted and EC50 values determined using GraphPad Prism software. Coefficients of variation ranged between 3 – 12% for the various treatment concentrations; n = 3. Error bars are omitted for visual clarity. The absences of signal in CP 55,940 treated NFAT Reporter cells (which do not express CB1R) confirms that the response observed in the CB1R Reporter Cells is specific to cannabinoid receptor function.INDIGO CB1R reporter assay antagonist dose-response analyses
CB1R reporter cells were co-treated with an EC80 concentration of the reference agonist CP 55,940 and varying concentrations of the CB1R reference antagonists CP 945,598, AVE-1625, AM251, Rimonabant, and AM6545. The range of determined IC50 values is shown. Treating the CB1R reporter cells with the calcineurin inhibitor FK-506 reaffirms that agonist-activated CB1R signals via the Ca+2 Calcineurin / NFAT signal transduction cascade. INDIGO’s Live Cell Multiplex (LCM) Assay confirmed that no treatment concentrations were cytotoxic (data not shown). Non-linear regression analyses of RLU vs. Log10[Antagonist, nM] were plotted and IC50 determinations made using GraphPad Prism software. Coefficients of variation ranged between 3 – 11% for the various treatment concentrations; n = 3. Error bars are omitted for visual clarity. All compounds were obtained from Cayman Chemical (Ann Arbor MI, USA).