| Product Description |
Recombinant CHO-K1 cells stably overexpress human hypocretin (orexin) receptor 2 (OX2) on the surface and contain high levels of G protein Gαq to couple with the receptor in downstream signaling pathways. |
| Applications |
Calcium mobilization assay, IP-One accumulation assay. |
| Expressed Gene |
NCBI reference sequence NM_001526; no expressed tags |
| Target Protein |
NP_001517 |
| Host Cell |
CHO-K1 |
| Size |
Two vials of frozen cells (>1×106 per vial in 1 mL) |
| Storage |
Store cells in liquid nitrogen immediately upon receipt. Thaw and recover cells within one year from the date received. |
| Mycoplasma Status |
Negative. The mycoplasma test was performed with MycoAlert™ PLUS Mycoplasma Detection Kit (Cat. No. LT07-318, Lonza). |
| Stability |
Stable through more than 16 passages with no significant changes in assay performance or expression profile. |
| Culture Properties |
Adherent |
| Freeze Medium |
45% Ham’s F-12K (Kaighn’s) (Cat. No. 21127, Life Technologies), 45% FBS (Cat. No. 10099-141, Gibco), 10% DMSO (Cat. No. D2650, Sigma) |
| Complete Growth Medium |
Ham’s F-12K (Kaighn’s), 10% FBS |
| Culture Medium |
Ham’s F-12K (Kaighn’s), 10% FBS, 400 μg/ml G418 (Cat. No. 10131-035, Gibco) |
| Gene Synonyms |
HCRTR2, ORXR2, OX2R, OXR2, |
| Background |
Hypocretin (orexin) receptor 2, also known as OX2, is a human protein encoded by the HCRTR2 gene. Orexin A and orexin B are neuropeptides originally identified as endogenous ligands for OX2. Orexin neuropeptides are produced by a small group of neurons in the lateral hypothalamic and perifornical areas, a region that is classically implicated with the control of mammalian feeding behavior. Orexin neurons that project throughout the central nervous system to nuclei are known to be important in the control of feeding, sleep-wakefulness, neuroendocrine homeostasis, and autonomic regulation. |
Figure 1. Orexin A-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/OX2 cells. The cells were loaded with Calcium-4 prior to stimulation with an OX2 receptor agonist, Orexin A. The intracellular calcium change was measured by FLIPRTETRA. The relative fluorescent units (RFU) were normalized and plotted against the log of the cumulative doses (5-fold dilution) of Orexin A (Mean ± SEM, n = 3). The EC50 of Orexin A on this cell was 1.36 nM.
Notes:
EC50 value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom) / (1+10^((LogEC50-X)*Hill Slope))
X is the logarithm of concentration. Y is the response
Y is % stimulation of RFU and starts at Bottom and goes to Top with a sigmoid shape.
Figure 2. Dose dependent stimulation of intracellular IP-One accumulation upon treatment with Orexin A in CHO-K1/OX2 cells. d2 acceptor fluorophore-labeled IP-One (Cat. No. 62IPAPEB; Revvity) and intracellular IP-One in CHO-K1/OX2 cells competitively bind with Europium Cryptate-labeled anti-IP-One antibody. The FRET signal decreases as the intracellular IP-One concentration rises and was measured by plate reader (Varioskan, Thermo). The EC50 of Orexin A on CHO-K1/OX2 cells was 4.15 nM.
For research use only. Not intended for human or animal clinical trials, therapeutic or diagnostic use.
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