| Assay Method Information | |
| | Antagonism Against Adrenoreceptors |
| Description: | Antagonism against the adrenoreceptor α1A was tested using a recombinant human α1A receptor CHO cell line which additionally also recombinantly expresses mtAeq (mitochondrial aequorin). Antagonism against the adrenoreceptor α2A was tested using a recombinant human α2A-Gα16 receptor fusion protein CHO cell line (PerkinElmer Life Sciences) which additionally also recombinantly expresses mtAeq. Antagonism against the adrenoreceptor α2B was tested using a recombinant human α2B receptor CHO cell line (PerkinElmer Life Sciences) which additionally also recombinantly expresses mtAeq. Antagonism against the adrenoreceptor α2C was tested using a recombinant human α2C receptor CHO cell line which additionally also recombinantly expresses a chimaric G protein (Gαqi3) and mtOb (mitochondrial obelin).The cells were cultivated at 37° C. and 5% CO2 in Dulbecco's modified Eagle's Medium/NUT mix F12 with L-glutamine which additionally contains 10% (v/v) inactivated foetal calf serum, 1 mM sodium pyruvate, 0.9 mM sodium bicarbonate, 50 U/ml penicillin, 50 μg/ml streptomycin, 2.5 μg/ml amphotericin B and 1 mg/ml Geneticin. The cells were passaged with enzyme-free Hank's-based cell dissociation buffer. All cell culture reagents used were from Invitrogen (Carlsbad, USA).Luminescence measurements were carried out on white 384-well microtitre plates. 2000 cells/well were plated in a volume of 25 μl and cultivated for one day at 30° C. and 5% CO2 in cell culture medium with coelenterazine (α2A and α2B: 5 μg/ml; α1a/c and α2C: 2.5 μg/ml). Serial dilutions of the test substances (10 μl) were added to the cells. After 5 minutes, noradrenaline was added to the cells (35 μl; final concentrations: 20 nM (α1a/c and α2C) or 200 nM (α2A and α2B)), and the emitted light was measured for 50 seconds using a CCD (charge-coupled device) camera (Hamamatsu Corporation, Shizuoka, Japan) in a light-tight box. The test substances were tested up to a maximum concentration of 10 μM. The IC50 values were calculated from the appropriate dose-response curves. |
| Affinity data for this assay | |
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