GENSCRIPT TM0442 Technical Manual

Human Recombinant κ-Opioid Receptor OPRK1 Stable Cell Line
Technical Manual No. TM0442 Version 06042010
I
Introduction…….…………………………………………………………………………….
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II
Background………………………………………………………………………………….
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III
Representative Data.………………………………………………………………………
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IV
Thawing and Subculturing.………………………………………………………………
2 V References ……………………………………………………………………………….
3 Limited Use License Agreement…………………………………………………………
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I. Introduction
Catalog Number: M00290 Cell Line Name: CHO-K1/OPRK1/Gα15
Gene Synonyms: OPRK1, OPRK, KOR-1, KOR Expressed Gene: Genbank Accession Number NM_000912; no expressed tags
Host Cell: CHO-K1/Gα15 Quantity: 2 vials (3×106 per vial) frozen cells Stability: 16 passages Application: Functional assay for OPRK1 receptor Freeze Medium: 45% culture medium, 45% FBS, 10% DMSO Complete Culture Medium: Ham’s F12, 10% FBS Culture Medium: Hams F12, 10% FBS, 100 μg/ml Hygromycin B, 200 μg/ml Zeocin Mycoplasma Status: Negative Storage: Liquid nitrogen immediately upon delivery.
II. Background
Opioid receptors and their endogenous peptide ligands play important roles in the reward and reinforcement of drugs such as heroin, cocaine, and alcohol. The κ-opioid receptor is a type of opioid receptor which binds the peptide opioid dynorphin as the primary endogenous ligand. κ-opioid receptors are widely distributed in the brain, spinal cord, and in pain neurons. They are associated with the risk for alcohol dependence.
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III. Representative Data
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CHO-K1/OPRK1/G15 CHO-K1/G15
EC50= 4.7 nM
Log[U-50488] M
RFU
Figure Intracellular calcium response from CHO-K1/Gα15 cells stably expressing human κ opioid receptor OPRK1
and from untransfected control cells. Cells were loaded with Calcium-4 then stimulated with the indicated concentrations of U-50488. Calcium responses were recorded on a FlexStation plate reader. Data represent the average +/- standard deviation of triplicate determinations.
IV. Thawing and Subculturing
Thawing: Protocol
1. Remove the vial from liquid nitrogen tank and thaw cells quickly in a 37°C water-bath.
2. Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol and transfer the cells to a 15 ml centrifuge tube containing 9 ml of complete growth medium.
3. Pellet cells by centrifugation at 200 x g force for 5 min, and discard the medium.
4. Resuspend the cells in complete growth medium.
5. Add 2 ml of the cell suspension per well in a 6 well-plate.
6. Add Hygromycin B and Zeocin to concentrations of 100 μg/ml and 200 μg/ml respectively the following day.
Subculturing: Protocol
1. Remove and discard culture medium.
2. Wash cells with PBS (pH=7.4) to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 ml of 0.05% (w/v) Trypsin- EDTA (GIBCO, Cat No. 25300) solution to 10 cm dish and observe the cells under an inverted microscope until cell layer is dispersed (usually within 3 to 5 minutes). Note: To avoid clumping, do not agitate the cells by hitting or shaking the dish while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting, centrifuge the cells 200 x g force for 5min, and discard the medium.
5. Resuspend the cells in complete growth medium with Hygromycin B and Zeocin and add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.
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Subcultivation Ratio: 1:3 to 1:8 weekly. Medium Renewal: Every 2 to 3 days
V. References
1. Zhu, J., Luo, L. Y., Li, J. G., Chen, C. and Liu-Chen, L. Y. (1997) Activation of the cloned human kappa opioid receptor by agonists enhances [35S]GTPgammaS binding to membranes: determination of potencies and efficacies of ligands, J. Pharmacol. Exp. Ther., 282, 676 - 684.
2. Chavkin, C., Sud, S., Jin, W., Stewart, J., Zjawiony, J. K., Siebert, D. J., Toth, B. A., Hufeisen, S. J. and Roth, B. L. (2004) Salvinorin A, an active component of the hallucinogenic sage salvia divinorum is a highly efficacious kappa-opioid receptor agonist: structural and functional considerations. J. Pharmacol. Exp. Ther., 308, 1197 - 1203.
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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:
1) 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.
2) 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.
3) 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 manufacture of such product. For information on the use of this product for other purposes, contact Marketing 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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