ChemiSCREEN™ CB1 Calcium-Optimized FLIPR Cell Line

ChemiSCREEN™ CB1 Calcium-Optimized FLIPR Cell Line

• ChemiScreen
• Chemicon (Millipore)

TRANSFECTION: Full-length human CB1 cDNA

CB1 is a GPCR that is expressed primarily in brain and nervous tissue, and mediates numerous CNS responses such as analgesia, appetite, cognition, memory and locomotor activity. A number of cannabinoid ligands bind to CB1 and activate Gi/o-mediated downstream responses, including inhibition of cAMP production and activation of ion channels and MAP kinases. Such ligands include exogenous agonists such as Delta9-THC, the main psychoactive component of the plant Cannabis sativa, and endogenous agonists such as anandamide that belong to eicosanoid family. A number of synthetic agonists such as CP55940 and R-(+)-WIN55212, and antagonists, such as SR141716A, for CB1 have been developed (Howlett et al., 2002). CB1 agonists have clinical utility in analgesia and antiemetic properties, whereas CB1 antagonists show promise for treatment of appetite in obesity disorders. Chemicon's cloned human CB1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CB1 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 agonists and antagonists of CB1.

Calcium flux assay, ligand binding assays

Human

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

GPCR Cell Lines

2 x 10(e6) cells/mL

2x10(e6) cells/vial

GROWTH MEDIA: 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

• CNR1
• CNR
• CANN6
• CB1A
• CB1
• CB1K5
• CB-R

Cell Line

Cells are frozen at 2 x 10(e6) cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma

GPCRs

RECOMMENDED ASSAY CONDITIONS: Cells are plated at ~50,000 cells per well in a 96-well clear bottom, black-walled assay plate in growth media lacking Genetecin/G418, and incubated overnight at 37°C, 5% CO2. On the day of the assay, loading media is prepared by mixing equal volumes Fluo-4 (1 mM stock in DMSO) and Pluronic F-127 (20% in DMSO), then diluting 1:500 in growth media (lacking Genetecin/G418, containing 2.5 mM probenecid), to achieve a final Fluo-4 concentration of 1 mM. Cells are incubated for 1 h in loading media at 37°C, 5% CO2, and washed 3 times, 200 mL/well each time, with Assay Buffer (HBSS with Ca++ and Mg++/20 mM HEPES/2.5 mM probenecid, added on the day of the assay). Assay Buffer is added (100 mL/well) and cells are returned to 37°C, 5% CO2. CB1 ligands were prepared as 5 mM stocks in 50% ethanol, and the 3x working stocks of the dilution series were prepared in prewarmed Assay Buffer containing 1% ethanol. Additions of 50 mL 3x working ligand stock to cells in 100 mL Assay Buffer were made with a Flex StationTM (Molecular Devices), and fluorescence was measured at ex. 485 nm, em. 525 nm.

Note: Cannabinoid ligands are poorly soluble in aqueous solution, and typically need to be dissolved in an organic solvent (DMSO or ethanol) before addition to assay buffer. However, such solvents can interfere with detection of intracellular calcium, typically at concentrations exceeding 1% DMSO or ethanol. In order to determine the optimal concentration of solvent to achieve effective ligand solubility with minimal solvent effect on calcium measurements, controls for solvent effects, containing solvent at concentrations equal to that in ligand solutions, should always be run in parallel with ligands.

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 37ºC 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 growth media, and replace media after 8-24 h. Cells are typically passaged 1:10 every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.