| Assay Method Information | |
| | Antagonistic Activity (IC50) of Compounds Against A2A Receptor |
| Description: | The IC50 (nM) values of the example compound HZ10126 and several reference compounds against the rat A2A receptor were measured in an in vitro assay, and the results are shown in Table 2. To be specific, each test compound was added to a 384-well plate Opti-Plate (PerkinElmer, 6007290) at a preset concentration and the wells were sealed with a sealing film. A 20 U A2A membrane (human adenosine A2A receptor membrane, PerkinElmer, RBHA2AM400UA) was added to 1 ml Assay Buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 1 μg/ml adenosine deaminase (Diazyme), 4° C.) to a final concentration of 25 nM, of which 50 μl was taken and added to the Opti-plate and incubated at 25° C. for 90 min. 100 μl of a 0.5% PEI solution was added to a UNIFILTER-96 GF/B filter plate (PerkinElmer, 6005177) to soak the filter plate at 4° C. for 90 min, and each well was washed twice with 500 μl Washing Buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl). The mixture in the Opti-plate was transferred to the UNIFILTER-96 GF/B filter plate, which was then washed 9 times with Washing Buffer (500 μl/well), and incubated at 55° C. for 10 min. 40 μl ULTIMA GOLD scintillation liquid (PerkinElmer, 77-16061) was added to each well, and the CPM (count per minute) value was read with TopCount (PerkinElmer, NTX). A series of concentrations were established for each compound and the CPM values were plotted versus the concentrations. IC50 values were calculated from the curve obtained. The reference compound is (3H)-CGS 21680, 250 μCi (30.7 Ci/mM) (PerkinElmer, NET1021). |
| Affinity data for this assay | |
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