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Human CRHR1-FLAG Stable Cell Line-HEK293T

Cat.No.: CSC-RG0235
Cell Line Description: HEK293T-HuCRHR1-FLAG cell line is a hypotriploid human cell line, which has been transfected with a Human corticotropin releasing hormone receptor 1 (CRHR1) tagged in the N-terminus with FLAG to allow stably express of the human CRHR1 tagged in the N-terminus with FLAG protein. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: Hypothalamic corticotropin releasing hormone (CRH) exerts its diverse physiological actions via cell surface G protein-coupled CRH receptors (CRHRs), of which two subtypes (CRHR1 and CRHR2) have been identified. CRH regulates pituitary ACTH secretion and mediates behavioral and autonomic responses to stress. Overproduction of CRH and stress system abnormalities are associated with psychiatric diseases such as depression, anxiety, eating disorders, and addiction. CRHR1 is expressed in pituitary corticotrophs and the brain. CRH binds to CRHR1 with high affinity, and activation of CRHR1 by CRH increases cAMP intracellular levels and activates protein kinase A. CRHR1 also couples to activation of MAPK as well as protein kinase C in an isoenzyme-specific manner. Selective CRHR1 antagonists offer new possibilities for the treatment of anxiety and depression.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293T
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human CRHR1.2. Protein expression: CRHR1 expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Corticotropin-Releasing Factor
Propagation: Complete growth medium: DMEM/F12, 10% FBS, 1 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 1:10 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: 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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