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.………………………………………………………………………

2

IV

Thawing and Subculturing….………………………………………………………………

2 V References ……………………………………………………………………………….

3 Limited Use License Agreement…………………………………………………………

4

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: Ham’s 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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0

50

100

150

200

250

CHO-K1/OPRK1/G 15
CHO-K1/G 15

EC50= 4.7 nM

Log[U-50488] M

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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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