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Human HTR2C/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0753
Cell Line Description: This human 5-HT2C-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant 5-HT2C expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between 5-HT2C and its ligands.
Background: 5-Hydroxytryptamine (5-HT, also commonly known as serotonin) is synthesized in enterochromaffin cells in the intestine and in serotonergic nerve terminals. In the periphery, 5-HT mediates gastrointestinal motility, platelet aggregation, and contraction of blood vessels. Many functions of the central nervous system are influenced by 5-HT, including sleep, motor activity, sensory perception, arousal and appetite. A family of 12 GPCRs and one ion channel mediate the biological effects of 5-HT. 5-HT2C, which couples to Gq in most cells to stimulate intracellular calcium, is prominently expressed in brain and appears to modulate depression, anxiety and appetite. The mRNA encoding 5-HT2C undergoes selective RNA editing that changes 4 amino acids in the second intracellular loop; these changes result in alteration of efficiency of coupling to G proteins. Alterations in editing of 5-HT2C have been detected in victims of suicidal depression and in mice treated with the SSRI, fluoxetine.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of HTR2C.2. Protein expression: HTR2C in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by Ro 60-0175: ~ 510 nM; EC50 for calcium mobilization by alpha-methyl-5-hydroxytryptamine: ~45 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: 5-Hydroxytryptamine
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: HTR2C 5-hydroxytryptamine (serotonin) receptor 2C, G protein-coupled [ Homo sapiens ]
Official Symbol: HTR2C
Synonyms: HTR2C; 5-hydroxytryptamine (serotonin) receptor 2C, G protein-coupled; 5 hydroxytryptamine (serotonin) receptor 2C , HTR1C; 5-hydroxytryptamine receptor 2C; 5 HT2C; 5-HT1C; 5-HT-1C; 5-HT-2C; serotonin receptor 2C; serotonin 5-HT-2C receptor; 5-hydroxytryptamine receptor 1C; HTR1C; 5-HT2C; 5-HTR2C;
Gene ID: 3358
mRNA Refseq: NM_000868
Protein Refseq: NP_000859
MIM: 312861
UniProt ID: P28335
Chromosome Location: Xq23
Pathway: Amine ligand-binding receptors, organism-specific biosystem; Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; 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;
Function: 1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine binding; G-protein coupled receptor activity; drug binding; phosphatidylinositol phospholipase C activity; receptor activity; serotonin binding; serotonin 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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