ChemiScreen™ M5 Calcium-Optimized FLIPR Cell Lines
Description:
ChemiScreen™ M5 Calcium-Optimized FLIPR Cell Lines
Trade Name:
ChemiScreen
Product Overview:
Full-length human CHRM5 cDNA, encoding M5
Background Information:
The muscarinic acetylcholine receptor family consists of five GPCRs that mediate some of the neurotransmission functions of acetylcholine in the CNS and the periphery. The M5 receptor, along with the M1 and M3 receptors, signal through Gq/11 and subsequent release of Ca++ from the ER (Caulfield and Birdsall, 1998). Although M5 is expressed in the CNS at relatively low levels, it appears to be the only muscarinic acetylcholine receptor expressed in midbrain dopaminergic neurons, and it has been shown to mediate dopamine release from these neurons. The M5 receptor is also expressed in blood vessels in the brain and the periphery, and studies with M5 knockout mice demonstrate that M5 mediates the vasodilatory action of acetylcholine on cerebral microvessels. Further studies with M5 knockout mice also indicate a role for M5 in the rewarding effects of cocaine and morphine (Wess, 2004). Chemicon's cloned human M5-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant M5 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between M5 and its ligands.
Application Notes:
Calcium flux assay, ligand binding assays
Usage Statement:
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.
Cell Line Type:
GPCR Cell Lines
Concentration:
2 x 10e6 cells/mL
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G protein, Gα15.
Packaging:
1 mL per vial
Materials Required but Not Delivered:
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
Cell Type:
Chem-1
Gene Symbol:
- CHRM5
- MGC41838
- HM5
Presentation:
Cells are frozen at 2 x 10e6 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Quality Assurance:
SPECIFICATIONS: EC50 for calcium mobilization by carbamoyl choline: ~ 19.5 nM
Protein or Enzyme Type:
GPCRs
Species:
Human
Storage Conditions:
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 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 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.