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| $12000.00 | |
| M01136 | |
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| Product Description | Recombinant CHO-K1 cells stably overexpress human receptor activity modifying protein 3 (RAMP3) and calcitonin receptor (CTR) on the surface. Amylin receptors (AMYR) are a class of special protein heterodimers, composed of a CTR combined with one of three homologous receptor activity-modifying proteins (RAMP1, RAMP2, RAMP3), forming three subtypes (AMY1, AMY2, AMY3). This cell contain high levels of G protein Gαs and luciferase to couple with the receptor in downstream signaling pathways. |
| Culture Properties | Adherent |
| Stability | Stable through more than 20 passages without significant changes in assay performance or expression profile. |
| 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. |
| Culture Medium | Ham’s F-12K (Kaighn’s), 10% FBS, 100 μg/ml Hygromycin B (Cat. No. 10687010, Life Technologies), 200 μg/ml Zeocin (Cat. No. R250-01, Life Technologies), 400 μg/ml Geneticin (Cat. No. 10131-027, Life Technologies) |
| Complete Growth Medium | Ham’s F-12K (Kaighn’s), 10% FBS |
| Freeze Medium-DATA | 45% Ham’s F-12 K (Kaighn’s) (Cat. No. 21127, Life Technologies), 45% FBS (Cat. No. 10099-141, Life Technologies), 10% DMSO (Cat. No. D2650, Sigma) |
Figure 1. Amylin-induced luciferase expression in CHO-K1/CRE-Luc/AMY3 cells. After stimulation by agonist rat amylin (Cat. No. HY-P1464, MCE), the cells are determined with Fire-Lumi™ Luciferase Assay System (Cat. No. L00877C-100, Genscript) and the relative luminescence units (RLU) were recorded by plate reader (Pherastar, BMG). The RLU were plotted against the log of the cumulative doses of rat amylin (Mean ± SEM, n = 3). The EC50 of rat amylin on cells was 0.37 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 RLU and starts at Bottom and goes to Top along a sigmoid curve.
»
Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with rat amylin in CHO-K1/CRE-Luc/AMY3 cells. d2 acceptor fluorophore-labeled cAMP (Cat. No. 62AM4PEB; Revvity) and intracellular cAMP in CHO-K1/CRE-Luc/AMY3 cells competitively bind with Europium Cryptate-labeled anti-cAMP antibody. The FRET signal decreases as the intracellular cAMP concentration rises and was measured by plate reader (Pherastar, BMG LabTech). The EC50 of rat amylin on CHO-K1/CRE-Luc/AMY3 cells was 1.33 nM. »
For research use only. Not intended for human or animal clinical trials, therapeutic or diagnostic use.
