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Human HRH1/Galpha15 Stable Cell Line-Chem-3

Cat.No.: CSC-RG0675
Cell Line Description: This human H1-expressing cell line is made in the Chem-3 host, which supports high levels of recombinant H1 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 H1 and its ligands.
Background: Histamine exerts its biological effects through a family of four GPCRs. Stimulation of the H1 histamine receptor causes smooth muscle contraction, induces vascular permeability, and stimulates catecholamine release from the adrenal medulla. H1 is also widely expressed in brain, and activation of H1 excites several types of neurons by blocking potassium conductance.
Growth Properties: Adherent
Host Cell: Chem-3
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of HRH1. 2. Protein expression: HRH1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by histamine: ~ 23 nM; IC50 for pyrilamine with 2xEC50 histamine: ~4.8 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Histamine
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: HRH1 histamine receptor H1 [ Homo sapiens ]
Official Symbol: HRH1
Synonyms: HRH1; histamine receptor H1; histamine H1 receptor; H1R; HH1R; histamine H receptor; histamine receptor, subclass H1; H1-R; hisH1;
Gene ID: 3269
mRNA Refseq: NM_000861
Protein Refseq: NP_000852
MIM: 600167
UniProt ID: P35367
Chromosome Location: 3p25
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: G-protein coupled receptor activity; histamine binding; histamine receptor activity; receptor activity; signal transducer activity;

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