ChemiScreen™ CCK2 Cholecystokinin Receptor Calcium-Optimized FLIPR Cell Lines

ChemiScreen™ CCK2 Cholecystokinin Receptor Calcium-Optimized FLIPR Cell Lines

ChemiScreen

Full-length human CCKB cDNA encoding CCK2

Cholecystokinins are a series of peptides of heterogeneous length (5 to 58 amino acids) that are derived from preprocholecystokinin and are found in gastrointestinal tissues and the central nervous system. Gastrin is a related peptide with 5 C-terminal amino acids identical to those of cholecystokinin. Two GPCRs, CCK1 (CCKA) and CCK2 (CCKB), bind to CCK and/or gastrin to mediate the biological effects of the peptides. CCK2 binds to CCK and gastrin with similar affinity, whereas CCK1 selectively binds sulfated CCK. Binding of ligands to CCK2 stimulates mobilization of intracellular calcium by activation of Gq/11. In the periphery, CCK2 is present in the stomach, where it mediates gastrin-stimulated gastric acid secretion. In the CNS, CCK2 has been implicated in anxiety, depression, schizophrenia, depression and opioid analgesia (Noble et al., 1999). Chemicon's cloned human CCK2-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CCK2 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 CCK2 and its ligands.

Calcium flux assay, ligand binding assays

Human

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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

• CCKBR
• CCK-BR
• CCK2R
• CCK-B
• GASR
• CCKRB
• CCK2-R

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 Gastrin I: ~ 3.1 nM
IC50 for YM 022: 22 nM
Signal:noise ratio at Gastrin I E_max: 623

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.