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Human HRH2/Galpha15 Stable Cell Line-Chem-2

Cat.No.: CSC-RG0676
Cell Line Description: This human H2-expressing cell line is made in the Chem-2 host, which supports high levels of H2 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to enhance coupling of the receptor to the calcium-signaling pathway. The cell line is an ideal tool for screening antagonists of interaction between H2 and its ligand.
Background: The histamine H2 receptor is a widely distributed G-protein coupled receptor, which is important in regulating a host of physiologic actions extending from gastric acid secretion to gastrointestinal motility. Histamine mediated activation of its receptor leads to signaling through both adenylate cyclase and phosphoinositide/protein kinase C second messenger systems. Antagonists for this receptor have proven to be effective therapy for acid peptic disorders of the GI tract. Certain antagonists are used in the treatment of neuropsychiatric and neurological diseases e.g. schizophrenia, Alzheimer"s disease and Parkinson"s disease.
Growth Properties: Adherent
Host Cell: Chem-2
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of HRH2. 2. Protein expression: HRH2 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by Amthamine: ~ 503.7 nM; IC50 for tiotidine with 2x EC50 concentration of amthamine: 31.5 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Histamine
Propagation: Complete growth medium: RPMI-1640 with 25 mM HEPES + 2 mM L-glutamine + 20% dialyzed heat-inactivated FBS + 1xPen-Strep + G-418 (400μg/mL) Plating medium: RPMI-1640 with 25 mM HEPES + 2 mM L-glutamine + 20% dialyzed heat-inactivated FBS + 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 containing 20 mL Plating Media (G418-free). Place the flask in a humidified incubator at 37°C with 5% CO2 for 16-24 h to allow cells to recover from thawing prior to centrifugation. 3. To remove residual DMSO, centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Plating Media (G418-free). Transfer to a T75 flask and place the flask in a humidified incubator at 37°C with 5% CO2.
Subculturing: 1. After 5-7 days, cells should be proliferating, and should be cultured in the presence of G418 to prevent loss of GPCR expression. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Growth Media. 2. Maintain the cells between 0.1 x 10^6 and 1 x 10^6 cells/mL. 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: 95% heat-inactivated FBS + 5% 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: HRH2 histamine receptor H2 [ Homo sapiens ]
Official Symbol: HRH2
Synonyms: HRH2; histamine receptor H2; histamine H2 receptor; HH2R; gastric receptor 1; gastric receptor I; H2R;
Gene ID: 3274
mRNA Refseq: NM_001131055
Protein Refseq: NP_001124527
MIM: 142703
UniProt ID: P25021
Chromosome Location: 5q35
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 (s) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem;
Function: G-protein coupled receptor activity; histamine receptor activity; receptor activity; signal transducer activity;

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