| Assay Method Information | |
| | Human Adenosine A2A Receptor Binding Assay |
| Description: | Test compound is weighed, dissolved in DMSO to make a stock solution of 10 mM, diluted with DMSO to prepare working solutions, then 100-fold diluted to the indicated concentrations. Human recombinant adenosine A2A receptors expressed in HEK-293 cells were used to prepare membranes in incubation buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 2 U/ml Adenosine Deaminase). Test compound or vehicle (2.2 μL) was added in 200 μL of membranes and incubated with 20 μL of 50 nM [3H] CGS-21680 for 90 minutes at 25 C. Non-specific binding was estimated in the presence of 50 μM NECA (5′-N-ethylcarboxamidoadenosine). The incubation was stopped by vacuum filtration onto 0.3% PEI (polyethylenimine) presoaked GF/B filters using a harvester followed by four washes with ice-cold 50 mM Tris-HCl, pH 7.4, and the radioactivity on GF/B filtermats counted in a scintillation counter (PerkinElmer TOPCOUNT ) to determine [3H] CGS-21680 specifically bound. Final concentration of vehicle DMSO is 1%.Data is fitted using the non-linear curve fitting routines in Meth-IQ software (ID Business Solutions Ltd., UK). IC50 is converted to Ki by Cheng-Prusoff equation: Ki=IC50/(1+[L]/KD) where [L] is concentration of radiolabeled ligand used in the assay. |
| Affinity data for this assay | |
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