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Human CNR2/Galpha15 Stable Cell Line-Chem-4

Cat.No.: CSC-RG0625
Cell Line Description: This human CB2 -expressing cell line is made in the Chem-4 host, which supports high levels of recombinant CB2 expression on the cell surface and contains high levels of promiscuous G proteins to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CB2 and its ligands.
Background: Cannabinoid compounds include exogenous drugs such as delta9-THC, the main psychoactive component of the plant Cannabis sativa, and endogenous mediators, such as anandamide, that belong to eicosanoid family.The biological effects of cannabinoids are mediated by a family of two Gi -coupled 7-transmembrane receptors, CB1 and CB2. The CB1 receptor is found primarily in brain and mediates the psychoactive effects of cannabinoid ligands. The CB2 receptor is expressed mainly in immune cells, including mast cells and CD40-activated B cells, where it mediates proliferation and inhibition of migration. Activation of CB2 inhibits the development of liver fibrosis. In bone, CB2 is expressed in both osteoblasts and osteoclasts, and functions to prevent bone loss. In addition, activation of CB2 has an antinociceptive effect in animal models of neuropathic, inflammatory, and acute pain; this effect is mediated by release of endogenous opioids in the periphery.
Growth Properties: Adherent
Host Cell: Chem-4
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of CNR2. 2. Protein expression: CNR2 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by CP55940: 78 nM; EC50 for calcium mobilization by WIN55, 212-2: 58 nM; EC50 for calcium mobilization by JWH 015: 603 nM; Signal/noise ratio: 963.
Application: Calcium flux assay, ligand binding assays
Sub-type: Cannabinoid
Propagation: Complete growth medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x Nonessential amino acids + 10mM HEPES + 1x Pen-Strep + 250μg/mL Genetecin/G-418 + 250μg/mL Hygromycin Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep Atmosphere: air, 95%; carbon dioxide (CO2), 5%Temperature: 37 °C
Starting Cells From Frozen Cell Stock: 1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years. 2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing Growth media. Place the flask in a humidified incubator at 37°C with 5% CO2. 3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
Subculturing: 1. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca2+ and Mg2+ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Growth media per 1 mL trypsin. 2. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Freezing medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 20% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep + 10% DMSO
Storage: Liquid nitrogen
Preservation: 1. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Subculture-Step 1). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10^6 cells/mL in Freezing Media (cell densities of 2-10 x 10^6 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at -70°C overnight. Store the vials in liquid nitrogen. 2. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Plating Media for plating for calcium assay.
Safety Considerations: The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.
Ship: Dry ice
Gene Name: CNR2 cannabinoid receptor 2 (macrophage) [ Homo sapiens ]
Official Symbol: CNR2
Synonyms: CNR2; cannabinoid receptor 2 (macrophage); cannabinoid receptor 2; CB2; testis-dominant CNR2 isoform CB2; CX5; CB-2;
Gene ID: 1269
mRNA Refseq: NM_001841
Protein Refseq: NP_001832
MIM: 605051
UniProt ID: P34972
Chromosome Location: 1p
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem; Neuroactive ligand-receptor interaction, conserved biosystem;
Function: cannabinoid receptor activity; receptor activity; signal transducer activity;

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C-fusion    N-fusion   Non-tagged
His    GST   Fc   Others
<1.0 eu/μg    <0.1 eu/μg   <0.01 eu/μg   Not required
Monomer Isolation    Dimer Isolation    Not required
>80% by SDS-PAGE    >90% by SDS-PAGE   >95% by SDS-PAGE   Others

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