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.

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III. Representative Data

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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.

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