GENSCRIPT TM0428 Technical Manual

Human Recombinant Corticotropin Releasing Factor Receptor CRF2
Stable Cell Line

Technical Manual No. TM0428 Version 06042010

I

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

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II

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

1

III

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

2

IV

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

3 V References ……………………………………………………………………………….

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

4

I. Introduction

Catalog Number: M00208
Cell Line Name: CHO-K1/CRF2/Gα15
Gene Synonyms: CRF2, CRHR2, CRFR2, CRH2
Expressed Gene: Genbank Accession Number NM_001883; no expressed tags
Host Cell: CHO-K1
Quantity: 2 vial (3×106 per vial) frozen cells
Stability: 16 passages
Application: Functional assay for CRF2 receptor
Freeze Medium: 45% culture medium, 45% FBS, 10% DMSO
Culture Medium: Ham’s F12, 10% FBS, 400 μg/ml G418, 200 μg/ml HygromycinB
Mycoplasma Status: Negative
Storage: Liquid nitrogen immediately upon delivery

II. Background

The corticotropin-releasing factor receptor 2 CRF2 is Gs-coupled GPCRs expressed in the brain, blood vessels
and intestine that bind to corticotropin-releasing factor (CRF). The CRF is a 41-amino acid peptide that plays an
important role in the integration of autonomic, neuroendocrine, and behavioral responses to stress. GenScript's
cloned human CRF2-expressing cell line co-expressing Gα15 is made in the CHO-K1 host.

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III. Representative Data

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0

10

20

30

40

50

60

70

CHO-K1/CRF2/G 15
CHO-K1

EC50= 45 nM

Log[Corticotro] M



RFU

Figure Intracellular calcium response from CHO-K1 cells stably expressing human CRF2 receptor and from

untransfected control cells. Cells were loaded with Calcium-4 then stimulated with the indicated concentrations of
corticotro. 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 G418 to concentrations of 200 μg/ml and 400 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 G418 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. Lovenberg et al., CRF2 alpha and CRF2 beta receptor mRNAs are differentially distributed between the rat
central nervous system and peripheral tissues (1995). Endocrinology. 136(9):4139-42

2. Bale, Vale, CRF and CRF receptors: role in stress responsivity and other behaviors (2004) Annu Rev
Pharmacol Toxicol. 2004;44:525-57. Review

GenScript USA Inc.

120 Centennial Ave., Piscataway, NJ 08854

Tel: [phone]

Fax: [phone]

Email: [e-mail]

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:

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: [phone]. Email: [e-mail].

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