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Human O3FAR1/Clytin Stable Cell Line-HEK293

Cat.No.: CSC-RG0659
Cell Line Description: These cells express a novel variant of clytin, a calcium-activated photoprotein, to enable sensitive luminescent detection of ligand-induced calcium flux. The clytin contains a mutation that increases its affinity for calcium to a level that permits detection of cytosolic calcium in many cells with greater sensitivity than other photoproteins targeted to the mitochondria. Luminescent calcium assays offer several advantages over fluorescent calcium assays including increased sensitivity and lack of interference from fluorescent compounds. These human GPR120 receptor-expressing cells were made by stable transfection of HEK293 cells with clytin and the GPR120 receptor. These stability-tested cells are ready for luminescent analysis of agonists, antagonists and modulators at the GPR120 receptor.
Background: It has been demonstrated that Omega-3 fatty acid administration to obese mice inhibited inflammation and enhanced insulin sensitivity, but these effects were absent in GPR120 knockout mice. GPR120 agonists may prove to be useful in suppression of chronic inflammation seen in obesity, which could reduce insulin resistance and help restore glucose control. A recent review suggests that the effects of marine n-3 fatty acids on inflammatory markers studied in healthy subjects, those at high risk for developing Cardiovascular Disease, and those with diagnosed Cardiovascular Disease are as yet not conclusive.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of O3FAR1.2. Protein expression: O3FAR1 in this cell line has been validated by immunocytochemical staining.
Application: Calcium Flux Assays
Sub-type: GPR
Propagation: Complete growth medium: DMEM/F12 with 2.5 mM glutamine, 10% heat-inactivated FBS, 1x Nonessential amino acids, 1x Pen-Strep, 250μg/mL Genetecin/G-418Plating medium: DMEM/F12 with 2.5 mM glutamine, 10% heat-inactivated FBS, 1x Nonessential amino acids, 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. 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 Accutase at 1 mL/T75 and keep at room temperature until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize Accutase by addition of 4 mL Growth Media per 1 mL Accutase. 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: 90% heat-inactivated FBS + 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: O3FAR1 omega-3 fatty acid receptor 1 [ Homo sapiens ]
Official Symbol: O3FAR1
Synonyms: O3FAR1; omega-3 fatty acid receptor 1; G protein coupled receptor 120 , G protein coupled receptor 129 , GPR120, GPR129; PGR4; G protein-coupled receptor 120; G protein-coupled receptor 129; G-protein coupled receptor 120; G-protein coupled receptor 129; G protein-coupled receptor PGR4; G-protein coupled receptor GT01; G-protein coupled receptor PGR4; G-protein-coupled receptor GT01; GT01; BMIQ10; GPR120; GPR129; MGC119984; DKFZp686F0824;
Gene ID: 338557
mRNA Refseq: NM_001195755
Protein Refseq: NP_001182684
MIM: 609044
UniProt ID: Q5NUL3
Chromosome Location: 10q23.33
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Free fatty acid receptors, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; Incretin Synthesis, Secretion, and Inactivation, organism-specific biosystem; Integration of energy metabolism, organism-specific biosystem; Metabolism, organism-specific biosystem; Regulation of Insulin Secretion, organism-specific biosystem;
Function: fatty acid binding; receptor activity; signal transducer activity; taste receptor activity;
Products Types ◆ Recombinant Protein

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