ChemiScreen™ α1a Adrenoceptor Calcium-Optimized FLIPR Cell Lines

ChemiScreen™ α1a Adrenoceptor Calcium-Optimized FLIPR Cell Lines

ChemiScreen

Full-length human ADRA1A transcript variant 1 cDNA encoding α1A

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 &alpha:- and β-adrenoceptors (Bylund et al., 1994). The three members of the α1 subclass of adrenoceptors, α1A, α1B and α1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue; the α1A subtype plays a prominent role in urogenital smooth muscle contraction and renal artery contraction (Hrometz et al., 1999; Ruffolo and Hieble, 1999). Activation of α1 adrenoceptors also influences cell proliferation; α1A inhibits cell growth by arresting progression at the G1/S transition (Shibata et al., 2003). The α1A subtype undergoes alternative splicing to generate four variants that differ at their C-termini, although these variants appear to be functionally identical (Chang et al., 1998). Chemicon's cloned human α1A-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant α1A 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 α1A and its ligands.

Calcium flux assay, ligand binding assays

Human

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ADRA1C, ADRA1L1, ALPHA1AAR

GPCR Cell Lines

2 x 10⁶ cells/mL

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

1 mL per vial

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

• ADRA1A
• ADRA1C
• ADRA1L1
• ALPHA1AAR
• ADRA1D

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.

SPECIFICATIONS: EC50 for calcium mobilization by A61603: ~ 2.3 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% CO₂. 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 Ca²⁺ and Mg²⁺-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.