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Human UTS2R/Galpha16 Stable Cell Line-Chem-2

Cat.No.: CSC-RG0766
Cell Line Description: This human GPR14-expressing cell line is made in the Chem-2 host, which supports high levels of recombinant GPR14 expression on the cell surface and contains high levels of the promiscuous G protein Galpha16 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between GPR14 and its ligands.
Background: Urotensin II, a cyclic 11-13 residue peptide expressed in motoneurons of the spinal cord, acts as a potent vasoconstrictor. The effects of urotensin II are mediated by binding to a GPCR, GPR14, which is expressed in endothelium, smooth muscle, heart and pancreas. Genetic deletion of GPR14 in mice renders aortae refractile to the contractile activity of urotensin II without changing baseline hemodynamics. Pharmacological inhibition of urotensin II/GPR14 interactions prevents renal insufficiency following renal artery ligation. Thus, GPR14 is an attractive target for a number of cardiovascular diseases.
Growth Properties: Adherent
Host Cell: Chem-2
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of UTS2R.2. Protein expression: UTS2R in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by Urotensin II ~ 1.7 nM; Z with 2x EC50 urotensin II: 0.54.
Application: Calcium flux assay, ligand binding assays
Sub-type: Urotensin
Propagation: Complete growth medium: RPMI-1640 with 25 mM HEPES + 2 mM L-glutamine + 20% dialyzed heat-inactivated FBS + 1xPen-Strep + G-418 (400μg/mL) Plating medium: RPMI-1640 with 25 mM HEPES + 2 mM L-glutamine + 20% dialyzed heat-inactivated FBS + 1x Pen-StrepAtmosphere: 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 containing 20 mL Plating Media (G418-free). Place the flask in a humidified incubator at 37°C with 5% CO2 for 16-24 h to allow cells to recover from thawing prior to centrifugation. 3. To remove residual DMSO, centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Plating Media (G418-free). Transfer to a T75 flask and place the flask in a humidified incubator at 37°C with 5% CO2.
Subculturing: 1. After 5-7 days, cells should be proliferating, and should be cultured in the presence of G418 to prevent loss of GPCR expression. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Growth Media. 2. Maintain the cells between 0.1 x 10^6 and 1 x 10^6 cells/mL. 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: 95% heat-inactivated FBS + 5% DMSO
Storage: Liquid nitrogen
Preservation: 1. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count cells. Centrifuge cells at 200 x g for 5 min. Resuspend cells at 2-5 x 10^6 cells/mL in Freezing Media. 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. Cells should be passaged at least once after thawing prior to use in calcium flux assays.
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: UTS2R urotensin 2 receptor [ Homo sapiens ]
Official Symbol: UTS2R
Synonyms: UTS2R; urotensin 2 receptor; G protein coupled receptor 14 , GPR14; urotensin-2 receptor; UR-II-R; urotensin II receptor; G protein-coupled receptor 14; G-protein coupled receptor 14; UTR; UTR2; GPR14; UR-2-R;
Gene ID: 2837
mRNA Refseq: NM_018949
Protein Refseq: NP_061822
MIM: 600896
UniProt ID: Q9UKP6
Chromosome Location: 17q25.3
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Other, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem; Neuroactive ligand-receptor interaction, conserved biosystem;
Function: G-protein coupled receptor activity; receptor activity; signal transducer activity; urotensin II receptor activity;

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