ChemiScreen™ Calcium-Optimized Stable Cell Line
Human Recombinant OX₂ Orexin Receptor

ChemiScreen™ Calcium-Optimized Stable Cell Line
Human Recombinant OX₂ Orexin Receptor

ChemiScreen

Full-length human HCRTR2 cDNA encoding OX₂

The orexin/hypocretin peptides orexin A and orexin B (also known as hcrt-1 and hcrt-2) are 33- and 28-amino acid peptides, respectively, that are preferentially expressed in hypothalamus. The orexins bind to two GPCRs, OX₁. and OX₂, to increase intracellular calcium. Orexins function to increase wakefulness, primarily through OX₂, which has been shown to harbor the causative mutation in narcoleptic dogs (Lin et al., 1999). In addition, the orexinergic system plays a role in feeding and stress responses (Kukkonen et al., 2002). Chemicon's cloned human OX₂-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant OX₂ 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 OX₂ and its ligands.

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 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

• HCRTR2
• Ox2r
• OX2R
• OTTHUMP00000039969

Cell Line

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 Orexin B: ~ 0.15 nM

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

TRANSFECTION: Full-length human HCRTR2 cDNA encoding OX₂ (Accession Number: NM_001526)

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

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 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.