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Human H3F3A/Galpha15 Stable Cell Line-Chem-3

Cat.No.: CSC-RG0677
Cell Line Description: This human H3-expressing cell line is made in the Chem-3 host, which supports high levels of recombinant H3 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 H3 and its ligands.
Background: The histamine H3 receptor was defined pharmacologically to be a presynaptic histamine receptor that binds selectively to R-(alpha)-methylhistamine and inhibits neurotransmitter release in the central and peripheral nervous systems. Molecular cloning of the H3 receptor confirmed it to be a Gi-coupled receptor widely expressed at high levels in the brain. H3 presents a potential therapeutic target for disorders of the central nervous system involving attention, cognition and feeding.
Growth Properties: Adherent
Host Cell: Chem-3
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of H3F3A.2. Protein expression: H3F3A in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by R-alpha-methylhistamine: ~ 6.5 nM; IC50 for iodophenpropit with 2xEC50 R-alpha-methylhistamine: ~1.8 nM.
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: H3F3A H3 histone, family 3A [ Homo sapiens ]
Official Symbol: H3F3A
Synonyms: H3F3A; H3 histone, family 3A; H3F3; histone H3.3; H3.3A; H3F3B; MGC87782; MGC87783;
Gene ID: 3020
mRNA Refseq: NM_002107
Protein Refseq: NP_002098
MIM: 601128
UniProt ID: P84243
Chromosome Location: 1q42.12
Pathway: Alcoholism, organism-specific biosystem; Alcoholism, conserved biosystem; Amyloids, organism-specific biosystem; Disease, organism-specific biosystem; Factors involved in megakaryocyte development and platelet production, organism-specific biosystem; Gene Expression, organism-specific biosystem; Hemostasis, organism-specific biosystem;

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