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Human TRHR/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0765
Cell Line Description: This human TRH-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant TRH 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 TRH and its ligands.
Background: Thyrotropin-releasing hormone (TRH) is a tripeptide (L-pyroglutamyl-L-histidinyl-L-proline amide) that is released from the hypothalamus to stimulate release of thyroid-stimulating hormone from the thyrotropes of the anterior pituitary. In addition, TRH is synthesized in other regions of the CNS, where it appears to function as a neuromodulator to influence arousal, cognition, motor activity, depression, and food and water intake. TRH functions in the autonomic nervous system to modulate gastrointestinal motility and acid secretion. The biological functions of TRH are mediated by binding to two Gq-coupled seven transmembrane receptors, TRH1 and TRH2. TRH1 is present in humans, rodents, and other vertebrates, whereas TRH2 has been identified only in rodents. Synthetic TRH (protirelin) is used clinically to evaluate the functionality of the hypothalamic-pituitary-thyroid axis.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of TRHR. 2. Protein expression: TRHR in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by TRH: ~4.8 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Thyrotropin Releasing Hormone
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: TRHR thyrotropin-releasing hormone receptor [ Homo sapiens ]
Official Symbol: TRHR
Synonyms: TRHR; thyrotropin-releasing hormone receptor; thyroliberin receptor; TRH-R; MGC141920;
Gene ID: 7201
mRNA Refseq: NM_003301
Protein Refseq: NP_003292
UniProt ID: P34981
Chromosome Location: 8
Pathway: 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; GPCRs, Class A Rhodopsin-like, organism-specific biosystem;
Function: receptor activity; signal transducer activity; thyrotropin-releasing hormone receptor 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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