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Human SUCNR1/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0761
Cell Line Description: This human GPR91-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant GPR91 expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at GPR91.
Background: GPR91 is a class A G-protein coupled receptor that binds selectively to succinate, an intermediate in the citric acid cycle, and couples to both the Gi/Go and Gq pathways. Renal proximal tubules are a major site of GPR91 expression, and succinate-mediated activation of renal GPR91 stimulates the release of renin to cause vasoconstriction and hypertension. In addition, GPR91 in retinal neurons mediates retinal vascularization induced by succinate, which accumulates in response to hypoxia. Dendritic cells also highly express GPR91, which mediates succinate-induced immune cell migration and activation in vitro and in vivo. Therefore, suppression of GPR91 is a potential strategy for treatment of vascular retinopathies, hypertension, and inflammation.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of SUCNR1.2. Protein expression: SUCNR1 in this cell line has been validated by immunocytochemical staining.
Application: Calcium flux assay, ligand binding assays
Sub-type: GPR
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 Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 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 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: SUCNR1 succinate receptor 1 [ Homo sapiens ]
Official Symbol: SUCNR1
Synonyms: SUCNR1; succinate receptor 1; G protein coupled receptor 91 , GPR91; P2Y purinoceptor 1; G protein-coupled receptor 91; G-protein coupled receptor 91; GPR91;
Gene ID: 56670
mRNA Refseq: NM_033050
Protein Refseq: NP_149039
MIM: 606381
UniProt ID: Q9BXA5
Chromosome Location: 3q25.1
Pathway: GPCRs, Class A Rhodopsin-like, organism-specific biosystem;
Function: G-protein coupled purinergic nucleotide receptor activity; G-protein coupled receptor activity; receptor activity; signal transducer activity;

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