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Human CRHR1/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0648
Cell Line Description: This cloned human CRF1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CXCR2 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 CRF1 and its ligands.
Background: The CRF1 receptor is a Gs-coupled GPCR expressed in the brain and pituitary gland that binds to several neuropeptides, including corticotropin-releasing factor (CRF) and urocortin, and the amphibian peptide sauvagine. CRF plays a predominant role in stress response mediated by the hypothalamic-pituitary-adrenal axis, and alterations in CRF and its receptors CRF1 and CRF2 appear to be linked to depression and anxiety. A number of small molecule antagonists of the CRF1 receptor have been characterized, including R121919, SC241, NBI27914, antalarmin, DMP-696, and CP 154,526. When delivered in animal models of psychiatric disorders, these antagonists display effectiveness in reducing stress-related behaviors.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of CRHR1.2. Protein expression: CRHR1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by Sauvagine: ~ 40 nM; IC50 for NBI27914: ~1.3 μM; IC50 for astressin: ~11 nM; Signal:noise at Sauvagine Emax: 340.
Application: Calcium flux assay, ligand binding assays
Sub-type: Corticotropin-Releasing Factor
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: CRHR1 corticotropin releasing hormone receptor 1 [ Homo sapiens ]
Official Symbol: CRHR1
Synonyms: CRHR1; corticotropin releasing hormone receptor 1; CRHR; corticotropin-releasing factor receptor 1; corticotropin releasing factor receptor; CRF R; CRF1; seven transmembrane helix receptor; corticotropin-releasing hormone receptor 1; corticotropin-releasing factor type 1 receptor; corticotropin releasing hormone receptor variant 1e; corticotropin releasing hormone receptor variant 1g; CRF-R; CRFR1; CRF-R1; CRFR-1; CRH-R1; CRHR1L; CRHR1f; CRF-R-1; CRH-R-1; CRH-R1h;
Gene ID: 1394
mRNA Refseq: NM_001145146
Protein Refseq: NP_001138618
MIM: 122561
UniProt ID: P34998
Chromosome Location: 17q12-q22
Pathway: Class B/2 (Secretin family receptors), organism-specific biosystem; G alpha (s) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class B Secretin-like, organism-specific biosystem; Long-term depression, organism-specific biosystem; Long-term depression, conserved biosystem;
Function: corticotrophin-releasing factor receptor activity; corticotropin-releasing hormone binding; peptide binding; protein binding; receptor activity; signal transducer activity;

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