GENSCRIPT TM0448 Technical Manual

Human Recombinant EP2 Prostanoid Receptor Stable Cell Line

Technical Manual No. TM0448 Version 10132010

I

Introduction ….…………………………………………………………………………….

1

II

Background………..……………………………………………………………………….

1

III

Representative Data………………………………………………………………………

2

IV

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

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

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

4

I. Introduction

Catalog Number: M00311
Cell Line Name: CHO-K1/EP2/Gα15
Gene Synonyms: PTGER2
Expressed Gene: Genbank Accession Number NM_000956; no expressed tags
Host Cell: CHO-K1/Gα15
Quantity: Two vials of frozen cells (3×106 per vial)
Stability: 16 passages
Application: Functional assay for EP2 receptor
Freeze Medium: 45% culture medium, 45% FBS, 10% DMSO
Complete Growth Medium: Ham’s F12, 10% FBS
Culture Medium: Ham’s F12, 10% FBS, 400 μg/ml G418, 100 μg/ml Hygromycin B
Mycoplasma Status: Negative
Storage: Liquid nitrogen immediately upon delivery

II. Background

Prostaglandin (PG) E2 exerts its actions by acting on a group of G-protein-coupled receptors (GPCRs). There are
four GPCRs responding to PGE2 designated subtypes EP1, EP2, EP3, and EP4 and multiple splicing isoforms of
the subtype EP3. The EP subtypes exhibit differences in signal transduction, tissue localization, and expression
regulation. Studies have identified that EP2 mediates many processes, such as facilitating ovulation and
fertilization, suppressing dendritic cell differentiation, and promoting amyloid-β formation in Alzheimer’s disease.

-1-

III. Representative Data

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4

0

20

40

60

80

100

CHO-K1/EP2/G 15

CHO-K1

EC50=21 nM
S/B = 6

Log[PGE2] M



RFU

Concentration-dependent stimulation of intracellular calcium mobilization by PGE2 in CHO-K1/EP2/Gα15
and CHO-K1 cells

Figure 1. PGE2-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-

K1/EP2/Gα15 and CHO-K1. The cells were loaded with Calcium-4 prior to stimulation with an EP2 receptor
agonist, PGE2. The intracellular calcium change was measured by FlexStation. The relative fluorescent units
(RFU) were plotted against the log of the cumulative doses (10-fold dilution) of PGE2 (Mean ± SD, n = 2). The
EC50 of PGE2 on EP2 co-expressing with Gα15 in CHO-K1 cells was 21 nM. The S/B of PGE2 on EP2 co­expressing with Gα15 in CHO-K1 cells was 6.

Notes:

1. EC

2. Signal to background Ratio (S/B) = Top/Bottom

value is calculated with four parameter logistic equation:

50

Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))
X is the logarithm of concentration. Y is the response
Y is RFU and starts at Bottom and goes to Top with a sigmoid shape.

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 10 ml of the cell suspension in a 10 cm dish.

6. Add Hygromycin B and G418 to concentrations of 100 μg/ml and 400 μg/ml respectively the following day.

-2-

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 culture medium and add appropriate aliquots of the cell suspension to new culture
vessels.

6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:3 to 1:8 weekly.
Medium Renewal: Every 2 to 3 days

V. References

1. Sugimoto Y, et al. (2007) Prostaglandin E receptors. J. Biol. Chem. 282(16):11613-11617

2. Regan JW. (2003) EP2 and EP4 prostanoid receptor signaling. Life Sci. 74(2-3):143-153.

3. Cimino PJ. (2008) Therapeutic targets in prostaglandin E2 signaling for neurologic disease. Curr. Med. Chem.
15(19):1863-1869.

GenScript USA Inc.

860 Centennial Ave., Piscataway, NJ 08854

Tel: [phone]

Fax: [phone]

Email: [e-mail]

Web: http://www.genscript.com

For Research Use Only.

-3-

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., 860 Centennial Avenue, Piscataway, New Jersey 08840, U.S.A. Phone: 1-732­885-9188. Fax: [phone]. Email: [e-mail].

-4-