| Product Description |
Recombinant CHO-K1 cells stably overexpress human melatonin receptor 1B (MT2) on the surface and contain high levels of G protein Gαi to couple with the receptor in downstream signaling pathways. |
| Applications |
Calcium flux assay, IP-One assay and cAMP accumulation assay. |
| Expressed Gene |
NCBI reference sequence NM_005959; no expressed tags |
| Target Protein |
NP_005950 |
| 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, Life Technologies), 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 Geneticin (Cat. No. 10131-035, Life Technologies), 100 μg/ml Hygromycin B (Cat. No. 10687010, Life Technologies) |
| Gene Synonyms |
MTNR1B, FGQTL2, MEL-1B-R, MT2 |
| Background |
Melatonin is a neuro hormone that plays a key role in the synchronisation of circadian and seasonal functions with cyclic environmental variations. In mammals, two melatonin receptors, MT1 and MT2, have been cloned. Activation of MT2 melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. |
Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with melatonin in CHO-K1/MT2/Gα15 cells. d2 acceptor fluorophore -labeled cAMP (Cat. No. 62AM4PEC; Revvity) and intracellular cAMP in CHO-K1/MT2/Gα15 cells competitively bind with Europium Cryptate-labeled anti-cAMP monoclonal antibody. The FRET signal decreases as the intracellular cAMP concentration rises and was measured by plate reader (Pherastar, BMG). The EC50 of melatonin on CHO-K1/MT2/Gα15 cells was 0.89 nM.
Figure 1. Melatonin-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/MT2/Gα15 cells. The cells were loaded with Calcium-4 (Cat. No. R8142; Molecular Devices) prior to stimulation with MT2 agonist, melatonin. The intracellular calcium change was measured by FLIPRTETRA. The relative fluorescent units (RFU) were recorded and normalized to plot against the log of the cumulative doses of melatonin (mean ± SEM, n = 3). The EC50 of melatonin on CHO-K1/MT2/Gα15 cells was 12.75 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 along a sigmoid curve.
For research use only. Not intended for human or animal clinical trials, therapeutic or diagnostic use.
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