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Human recombinant h4 histamine receptor stable cell line

Human Recombinant H4 Histamine Receptor Stable Cell Line

Technical Manual No. TM0439 Version 10132010
Introduction ….……………………………………………………………………………. Background…………………………………………………………………………………. Representative Data……………………………………………………………………… Thawing and Subculturing……………………………………………………………… 2 References ………………………………………………………………………………. Limited Use License Agreement………………………………………………………… Introduction

Gene Synonyms: GPRv53, HH4R, GPCR105, AXOR35, SP9144 Expressed Gene: Genbank Accession Number NM_021624; no expressed tags Quantity: Two vials of frozen cells (3×106 per vial) Application: Functional assay for H4 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, 100 μg/ml Hygromycin B, 400 μg/ml G418 Storage: Liquid nitrogen immediately upon delivery Background

Histamine receptor family consists of four members, namely, H1, H2, H3, and H4. The histamine H4 receptor is the most recently identified G protein-coupled histamine receptor that binds to several neuroactive drugs, including amitriptyline and clozapine. So far, H4 receptors have been found only on haematopoietic cells. This signifies its important role as a drug target for the treatment of inflammatory diseases. Representative Data
Concentration-dependent stimulation of intracellular calcium mobilization by Histamine in CHO-
K1/H4/Gα15 and CHO-K1/Gα15 cells
EC50 = 147 nM
CHO-K1/H4/G15
CHO-K1/G15
U
RF
100
-12 -11 -10
Log[Histamine] M
Figure 1. Histamine-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-
K1/H4/Gα15 and CHO-K1/Gα15 cells. The cells were loaded with Calcium-4 prior to stimulation with a H4 receptor
agonist, Histamine. 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 Histamine (Mean ± SD, n = 2). The
EC50 of Histamine on H4 co-expressing with Gα15 in CHO-K1 cells was 147 nM. The S/B of Histamine on H4 co-
expressing with Gα15 in CHO-K1 cells was 39.
Notes:
1. EC50 value is calculated with four parameter logistic equation: 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. Signal to background Ratio (S/B) = Top/Bottom Thawing and Subculturing
Remove the vial from liquid nitrogen tank and thaw cells quickly in a 37°C water-bath. 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. Pellet cells by centrifugation at 200 x g force for 5 min, and discard the medium. Resuspend the cells in complete growth medium. Add 10 ml of the cell suspension in a 10 cm dish. Add Hygromycin B and G418 to concentrations of 100 μg/ml and 400 μg/ml respectively the following day. Wash cells with PBS (pH=7.4) to remove all traces of serum that contains trypsin inhibitor. 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. 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. Resuspend the cells in culture medium and add appropriate aliquots of the cell suspension to new culture vessels. Subcultivation Ratio: 1:3 to 1:8 weekly. Medium Renewal: Every 2 to 3 days References
Leurs R, Chazot PL, Shenton FC, et al. (2009) Molecular and biochemical pharmacology of the histamine H4 receptor. J. Pharmacol. 157(1):14-23. Lim HD, van Rijn RM, Ling P, et al. (2005) Evaluation of histamine H1-, H2-, and H3-receptor ligands at the human histamine H4 receptor: identification of 4-methylhistamine as the first potent and selective H4 receptor agonist., J. Pharmacol. Exp. Ther. 314(3):1310-21. 860 Centennial Ave., Piscataway, NJ 08854
For Research Use Only.

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. 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. The products or its components are used for research purposes only. 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 purposes other than research, contact Marketing Department, GenScript USA Inc., 860 Centennial Avenue, Piscataway, New Jersey 08854, U.S.A. Phone: 1-732-885-9188. Fax: 1-732-210-0262. Email:

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