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Human GRM3-FLAG Stable Cell Line-HEK293T Galphaqi5

Cat.No.: CSC-RG0304
Cell Line Description: HEK293T Galphaqi5-HuGRM3-FLAG cell line is a hypotriploid human cell line, which has been transfected with a human glutamate receptor, metabotropic 3 (GRM3) tagged in the N-terminus with FLAG to allow stably express of the human GRM3 tagged in the N-terminus with FLAG. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: Metabotropic glutamate receptor mGluR3 is a G protein-coupled receptor for glutamate. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and functions to regulate excitability via pre- and postsynaptic mechanisms. The mGluR3 receptor is widely expressed in glial cells but also shows discrete localization both pre- and postsynaptic on glutamatergic and other neurotransmitter synapses. It is also expressed within forebrain regions including hippocampus and thalamus. Agonists of mGluR3 have been shown to suppress enhanced glutamatergic excitations in brain synapses known to be involved in the expression of fear/anxiety in animals and humans. It is anticipated that mGluR3 receptor represent a promising new target for treatment of anxiety and stress-related disorders in humans.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293T Galphaqi5
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human GRM3.2. Protein expression: GRM3 expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Glutamate
Propagation: Complete growth medium: DMEM with GlutaMAX (Gibco 10566), 10% FBS (dialyzed), 2 mM sodium pyruvate, 10 μg/mL puromycinAtmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Starting Cells From Frozen Cell Stock: 1. Remove the packaging cell lines from liquid nitrogen and carry out a quick thaw. Float the cells in the 37°C water bath for 2 minutes until nearly (80%) thawed. Once cells are thawed, it is important to dilute the cells 1:10 in growth media immediately to reduce the potentially toxic effects of the DMSO preservative on the cells.2. Clean the outside of the vial with 70% ethanol before opening.3. Remove the cells from the vial and add slowly into a 15ml conical tube containing 10 ml pre-warmed media. 4. Centrifuge for 3 minutes 1000 xg to pellet cells and remove the supernatant.5. Add 14 ml of media and transfer cells to a T25 flask or a 100 mm culture dish.6. Place the cells in the 37°C incubator with 5% CO2.7. Allow incubation for 3-4 days to reach confluence. The cells will re-attach to the surface over a period of several days in culture at 37°C.
Subculturing: 1. Remove and discard culture medium.2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.5. Add appropriate aliquots of the cell suspension to new culture vessels.Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 weekly is recommended Medium Renewal: 2 to 3 times per week
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 90%; DMSO, 10%
Storage: Liquid nitrogen
Preservation: 1. Detach cells from culture dish according to the Sub-Culture Procedure.2. Resuspend cells at a density of 5 x 10^6 cells/mL in freeze medium.Note: A T-75 culture flask typically yields enough cells for preparing two frozen vials.3. Aliquot 1 mL cells into cryogenic vials.4. Place vials in a freezing container and store at –80 °C overnight.5. Transfer vials to liquid nitrogen for long term storage. If properly stored, cells should remain stable for years.
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: GRM3 glutamate receptor, metabotropic 3 [ Homo sapiens ]
Official Symbol: GRM3
Synonyms: GRM3; glutamate receptor, metabotropic 3; metabotropic glutamate receptor 3; GPRC1C; mGlu3; MGLUR3; glutamate metabotropic receptor 3; GLUR3;
Gene ID: 2913
mRNA Refseq: NM_000840
Protein Refseq: NP_000831
MIM: 601115
UniProt ID: Q14832
Chromosome Location: 7q21.1-q21.2
Pathway: Class C/3 (Metabotropic glutamate/pheromone receptors), organism-specific biosystem; Cocaine addiction, organism-specific biosystem; Cocaine addiction, conserved biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class C Metabotropic glutamate, pheromone, organism-specific biosystem; Glutamatergic synapse, organism-specific biosystem; Glutamatergic synapse, conserved biosystem;
Function: G-protein coupled receptor activity; G-protein coupled receptor activity; calcium channel regulator activity; glutamate receptor activity; group II metabotropic glutamate receptor activity; receptor activity; signal transducer activity;

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Optional requirements on this protein    +Expand
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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