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Human HTR1A/Galpha15 Stable Cell Line-Chem-3

Cat.No.: CSC-RG0750
Cell Line Description: This human 5-HT1A-expressing cell line is made in the Chem-3 host, which supports high levels of recombinant 5-HT1A 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-HT1A 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-HT1A has been implicated in anxiety and depression. Desensitization of 5-HT1A has been correlated with the functional improvements in depression observed 2-3 weeks after initiation of treatment with SSRIs. Decreased 5-HT1A is observed in anxiety disorders, and mice lacking 5-HT1A display increased anxiety. Buspirone, a partial agonist for 5-HT1A , is used clinically for treatment of anxiety disorders.
Growth Properties: Adherent
Host Cell: Chem-3
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of HTR1A.2. Protein expression: HTR1A in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by 8-hydroxy-DPAT: ~ 8.8 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: 5-Hydroxytryptamine
Propagation: Complete growth medium: RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate + 10% heat-inactivated FBS + 100 U/ml penicillin + 100 U/ml streptomycin + 1% Chem-3 Growth Supplement + G-418/Geneticin (600ug/mL)Note: The Chem-3 Growth Supplement is required for growth of Chem-3 cell linesPlating medium: RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate + 10% heat-inactivated FBS + 100 U/ml penicillin + 100 U/ml streptomycinAtmosphere: 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 conical tube containing 10 mL Growth Media. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Growth Media. Transfer to a T75 flask and place the flask in a humidified incubator at 37 degree °C with 5% CO2.
Subculturing: 1. Maintain the cells in suspension between 0.1 x 10^6 and 1 x 10^6 cells/mL. 2. Cells are typically passaged 1:10 every 2-3 days, or 1:20 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator. Chem-3 cells tend to proliferate rapidly, so care should be taken not to allow them to become overconfluent.
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Freezing medium: RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate + 20% 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 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: HTR1A 5-hydroxytryptamine (serotonin) receptor 1A, G protein-coupled [ Homo sapiens ]
Official Symbol: HTR1A
Synonyms: HTR1A; 5-hydroxytryptamine (serotonin) receptor 1A, G protein-coupled; 5 hydroxytryptamine (serotonin) receptor 1A , ADRB2RL1, ADRBRL1; 5-hydroxytryptamine receptor 1A; 5 HT1A; 5-HT1a receptor; serotonin receptor 1A; G protein coupled receptor; guanine nucleotide-binding regulatory protein-coupled receptor; G-21; 5HT1a; 5-HT1A; 5-HT-1A; ADRBRL1; ADRB2RL1;
Gene ID: 3350
mRNA Refseq: NM_000524
Protein Refseq: NP_000515
MIM: 109760
UniProt ID: P08908
Chromosome Location: 5q11.2-q13
Pathway: Amine ligand-binding receptors, organism-specific biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; Monoamine GPCRs, organism-specific biosystem;
Function: G-protein coupled receptor activity; drug binding; receptor activity; serotonin binding; serotonin receptor activity; signal transducer activity;

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