Human Melanin Concentrating Hormone Receptor 2 Stable Cell
Line
Technical Manual No. TM0295
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Version 06042010
Introduction ….…………………………………………………………………………….
Background………..……………………………………………………………………….
Representative Data….……………………………………………………………………
References ……………………………………………………………………………….
Limited Use License Agreement …………………………………………………………
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Introduction
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Catalog Number: M00127
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Cell Line Name: MCHR2
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Description:
The MCHR2 reporter cell line is created by transfection of pcDNA3.1/MCHR2 in HEK293 cell lines. The
transfected cells are stably selected by 150 µg/ml G418. Single-cell clones with high MCHR2 inducibility
are isolated using ring cloning and serial dilution. The clones with the largest dynamic ranges in calcium
influx activity are chosen for pharmacological and stability studies.
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Applications: Functional assay for MCH receptor 2
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Quantity: 2 vial (2 x 106) frozen cells
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Host Cell: HEK293
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Cell Phenotype: Adherent/epithelial
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Mycoplasma: Negative
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Storage: Liquid nitrogen immediately upon delivery
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Culture Medium: DMEM, 10% FBS, 700 µg/ml G418.
Background:
Melanin-concentrating hormone receptor 2 (MCHR2) is one member of the neuropeptide receptor group. MCHR2
is expressed in the frontal lobe, temporal lobe, occipital lobe, precentral gyrus, pons, hippocampus, amygdala,
putamen, and caudate nucleus, with relatively high levels in the cerebral cortex. Because MCHR2 is specially
expressed in humans, its functional has not yet been identified. The proposed roles for MCH-R2 are mediation of
the cognitive and emotional representation of food intake and involvement in the regulation of nutritional
homeostasis by MCH. This manual describes establishment of a cell line and a protocol of pharmacologically
validated human MCHR2 GPCR receptor (Genebank Accession Number: NM_032503)
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Representative Data
MCH Dose Response of HEK293/MCHR2 Cell Line
Area under the ground
1000000.0
750000.0
500000.0
EC50=3.23 nm
250000.0
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Log[MCH] mM
1. 12-24 hours before running the assay, seed cells at a density of 3×104 to 5×104 cells/well in a 96-well
black plate.
2. The day of the assay, cells should be 100% confluent. Remove cell plates from the incubator. Do not
remove the supernatant. Add an equal volume of Fluo3 Loading Buffer to each well (100 µL per well for
96-well plates, 25 µL per well for 384 well plates)
3. Incubate the plate at 37°C in the dark for one hour.
4. Prepare agonist addition plates in advance of assay. To a 96-well plate, add 10x working concentration of
agonist compound in Calcium Assay 25 µl solution A, and add 25 µl/well to cell plate.
5. Read with FlexStation using the specified settings and save data. The assay should be completed within
three to five minutes after addition, but we recommend collecting data for a minimum of six minutes
during assay development. Usually 90 seconds are enough.
Instrument Optical Parameters
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Excitation wavelength (nm)
485
Emission wavelength (nm)
525
Emission cut-off (nm)
515
References
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Songzhu An et al. Identification and characterization of a melanin concentrating hormone receptor. PNAS,
2001, 98:7576-81.
Jeffrey Hill et al. Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor.
JBC, 2001, 276:20125-129.
Wang S. Identification and pharmacological characterization of a novel human melanin-concentrating
hormone receptor, MCH-R2. J Biol Chem, 17:17 (2001).
Rodriguez M. Cloning and molecular characterization of the novel human melanin concentrating hormone
receptor mch2. Mol Pharmacol, 2001, 60(4):632-9.
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Tel: 732-885-9188, 732-885-9688
Fax: 732-210-0262, 732-885-5878
Email: info@genscript.com
Web: http://www.genscript.com
For Research Use Only.
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Limited Use License Agreement
This is a legal agreement between you (Licensee) and GenScript USA Inc. governing use of GenScript's stable cell
line products and protocols provided to licensee. By purchasing and using the stable cell line, the buyer agrees to
comply with the following terms and conditions of this label license and recognizes and agrees to such restrictions:
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2)
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The products are not transferable and will be used at the site where they were purchased. Transfer to another
site owned by buyer will be permitted only upon written request by buyer followed by subsequent written
approval by GenScript.
The purchaser cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using
this product or its components to a third party.
The products sold by GenScript are for laboratory and animal research purposes only. The products are not to
be used on humans, for consumption, or for any unlawful uses.
GenScript USA Inc. will not assert against the buyer a claim of infringement of patents owned or controlled by
GenScript USA Inc. and claiming this product based upon the manufacture, use or sale of a clinical diagnostic,
therapeutic and vaccine, or prophylactic product developed in research by the buyer in which this product or its
components has been employed, provided that neither this product nor any of its components was used in the
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Department, GenScript USA Inc., 120 Centennial Avenue, Piscataway, New Jersey 08840, U.S.A. Phone: 1-732885-9188. Fax: 1-732-210-0262. Email: marketing@genscript.com.
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