Assay Method Information

Assay Name:  Binding Assays
Description:  [3H]R N6-Phenylisopropyladenosine (40, [3H]R-PIA, 63 Ci/mmol), [3H](2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine) (41, [3H]CGS21680, 40.5 Ci/mmol) and [25I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (42, [125I]I-AB-MECA, 2200 Ci/mmol) were purchased from Perkin-Elmer Life and Analytical Science (Boston, Mass.). Test compounds were prepared as 5 mM stock solutions in DMSO and stored frozen. Pharmacological standards 1b (A3AR agonist), adenosine-5′-N-ethylcarboxamide (43, NECA, nonselective AR agonist) and 2-chloro-N6-cyclopentyladenosine (44, CCPA, A1AR agonist) were purchased from Tocris R&D Systems (Minneapolis, Minn.).Cell Culture and Membrane Preparation CHO cells stably expressing the recombinant hA1 and hA3ARs and HEK293 cells stably expressing the hA2AAR were cultured in Dulbecco's modified Eagle medium (DMEM) and F12 (1:1) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 μmol/mL glutamine. In addition, 800 μg/mL geneticin was added to the A2A media, while 500 μg/mL hygromycin was added to the A1 and A3 media. After harvesting, cells were homogenized and suspended in PBS. Cells were then centrifuged at 240 g for 5 min, and the pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2. The suspension was homogenized and was then ultra-centrifuged at 14,330 g for 30 min at 4° C. The resultant pellets were resuspended in Tris buffer, incubated with adenosine deaminase (3 units/mL) for 30 min at 37° C. The suspension was homogenized with an electric homogenizer for 10 sec, pipetted into 1 mL vials and then stored at −80° C. until the binding experiments. The protein concentration was measured using the BCA Protein Assay Kit from Pierce Biotechnology, Inc. (Rockford, Ill.).27Binding Assays:Into each tube in the binding assay was added 50 μL of increasing concentrations of the test ligand in Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2, 50 μL of the appropriate agonist radioligand, and finally 100 μL of membrane suspension. For the A1AR (22 μg of protein/tube) the radioligand used was [3H]40 (final concentration of 3.5 nM). For the A2AAR (20 μg/tube) the radioligand used was [3H]41 (10 nM). For the A3AR (21 μg/tube) the radioligand used was [125I]42 (0.34 nM). Nonspecific binding was determined using a final concentration of 10 μM 43 diluted with the buffer. The mixtures were incubated at 25° C. for 60 min in a shaking water bath. Binding reactions were terminated by filtration through Brandel GF/B filters under a reduced pressure using a M-24 cell harvester (Brandel, Gaithersburg, Md.). Filters were washed three times with 3 mL of 50 mM ice-cold Tris-HCl buffer (pH 7.5). Filters for A1 and A2AAR binding were placed in scintillation vials containing 5 mL of Hydrofluor scintillation buffer and counted using a Perkin Elmer Liquid Scintillation Analyzer (Tri-Carb 2810TR). Filters for A3AR binding were counted using a Packard Cobra II γ-counter. The Ki values were determined using GraphPad Prism for all assays.Similar competition binding assays were conducted using HEK293 cell membranes expressing mARs using [125I]42 to label A1 or A3ARs and [3H]41 to label A2AARs. IC50 values were converted to Ki values as described.28 Nonspecific binding was determined in the presence of 100 μM 43.
Affinity data for this assay
 

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