ChemiScreen™ Human Recombinant β₁ Adrenoceptor Calcium-Optimized Stable Cell Line

ChemiScreen™ Human Recombinant β₁ Adrenoceptor Calcium-Optimized Stable Cell Line

ChemiScreen

Full-length human ADRB1 cDNA encoding β₁

The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenergic receptors (Bylund et al., 1994). The three members of the β-adrenergic receptor family, β₁, β₂ and β₃, couple to Gs to increase cAMP upon activation. In the heart, the β₁ receptor constitutes 70-80% of the β -adrenergic receptors. Activation of cardiac β-adrenergic receptors, acutely increases heart rate, cardiac output, and cardiac automaticity, and chronically increases cardiac myocyte apoptosis. In failing hearts, the β1 subtype is downregulated and desenstitized, probably as a result of increased catecholamine levels. As a result, β-adrenergic receptor antagonists (β blockers) are effective in the treatment of congestive heart failure and arrhythmia (Lohse et al., 2003). Chemicon's cloned human β1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant β1 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between the β₁ and its ligands.

Calcium flux assay, ligand binding assays

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GPCR Cell Lines

2 x 10⁶ cells/mL

Chem-1, an adherent cell line expressing the promiscuous G protein, Gα15.

DMEM containing 4.5 g/L glucose/10% heat inactivated fetal bovine serum/1x nonessential amino acids/10 mM HEPES/0.25 mg/ml Geneticin (G418)/100 U/ml each penicillin and streptomycin

Chem-1

• ADRB1
• ADRB1R
• RHR
• B1AR
• BETA1AR

Cells are frozen at 2 x 10⁶ cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

EC50 for calcium mobilization by Denopamine: ~ 0.65 nM
EC50 for calcium mobilization by Xamoterol: ~ 6.0 nM

GPCRs

Human

Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years. 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 20 mL growth media, and place in a humidified 37°C incubator with 5% CO<LB/>SUB.2</sub>. After 8-24 h, cells will adhere to the plate, at which time the media should be replaced to remove residual DMSO. Cells are passaged by washing with Ca⁺⁺ and Mg⁺⁺-free HBSS (10 mL/T75), incubating with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at 37°C, and rapping the side of the flask to dislodge the cells. Neutralize the trypsin by addition of 4 volumes growth media. Cells are typically passaged 1:10 with every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.