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.
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Human Recombinant H4 Histamine Receptor Stable Cell Line 
Representative Data 
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 
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