| Product Description |
Recombinant CHO-K1 cells stably overexpress human adenosine A2B receptor (ADORA2B) on the surface and contain high levels of G protein Gαs to couple with the receptor in downstream signaling pathways. |
| Applications |
Calcium flux assay, cAMP accumulation assay and IP-One assay. |
| Expressed Gene |
NCBI reference sequence NM_000676; no expressed tags |
| Target Protein |
NP_000667 |
| 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 |
A2bR, ADORA2 |
| Background |
The adenosine receptors ADORA2B is Gs and Gq/G11-coupled GPCR expressed in the large intestine, cecum, and urinary bladder. ADORA2B receptor mediates relaxation to adenosine in human small coronary arteries which is independent of NO but dependents in part on a K+-sensitive mechanism. Pharmacological or molecular biologic activation of ADORA2B receptor may prevent glomerular remodeling associated with glomerulosclerosis, renal disease, and abnormal growth associated with hypertension and diabetes. |
Figure 1: NECA-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/ADORA2B/Gα15 cells. The cells were loaded with Calcium-4 (Cat. No. R8142; Molecular Devices) prior to stimulation with ADORA2B agonist, NECA. 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 NECA (mean ± SEM, n = 3). The EC50 of NECA on CHO-K1/ADORA2B/Gα15 cells was 0.24 μM.
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.
Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with NECA in CHO-K1/ADORA2B/Gα15 cells. d2 acceptor fluorophore -labeled cAMP (Cat. No. 62AM4PEC; Revvity) and intracellular cAMP in CHO-K1/ADORA2B/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 NECA on CHO-K1/ADORA2B/Gα15 cells was 15.58 nM.
For research use only. Not intended for human or animal clinical trials, therapeutic or diagnostic use.
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