Assay method adenosine receptors, the efficacy and selectivity t of compounds different subtypes of adenosine receptors in human determine the following tests were used. Determination of A1 receptor binding: Chinese hamster ovary cells, the human A1 receptors were placed in a medium with 10% f 12 Nut.Mix.F fetal K calf serum, 2 mM L-glutamine and 200 mgmL 1 geneticin, bred. Confluent cells were scraped from the culture flasks and centrifuged at 1500 g for 5 min. The pellet was homogenized Evodiamine in a glass homogenizer and centrifuged at 40,000 g for 25 min. The final pellet was resuspended in assay buffer. The radioligand was 8 1.3 cyclopentyl dipropylxanthine and increasing concentrations of test compounds to the resulting Membranpr Paration added and for 90 min at room temperature. The samples collected on glass filter was added to scintillation fluid and Z hlungen Per minute were recorded on a Packard TopCount.
A1 receptor functional assay: This test measures the ability to inhibit F antagonists induced A1 I GTPgS AB MECA by binding to cell membranes. Geldanamycin The test was performed in a white S Fl Che binding 96-well Optiplate. Assay components were added as follows: 25 ml of assay buffer, 25 ml of 10 mM GDP, 25 ml 1.25 GTPgS nM, 100 nM, 25 ml of I AB MECA and 25 ml of increasing concentrations of compound or vehicle. The membranes were diluted in assay buffer to 25 mgmL 1 and added with 50 ml of WGA-SPA beads and to each well. After incubation at room temperature for 90 minutes at 850 g the plate was centrifuged for 10 min, and immediately read on a Packard TopCount. Determination of A2a receptor binding: A2a HEK 293 membranes were suspended in assay buffer.
The radioligand, ZM241385 and increasing concentrations of test compounds were added to the membrane preparation and incubated for 60 minutes at room temperature. The samples collected on glass filter was added to scintillation fluid and Z hlungen Per minute were recorded on a Packard TopCount. A2b receptor function test: A reporter gene assay using Chinese hamster ovary expressing both a reporter luciferase and functional human A2b adenosine receptor was used, transfected. The cells were grown to confluence in Dulbecco’s minimum essential medium with 10% f Fetal K Calf serum, glutamine 2mML, 0.4 mgmL 1 L proline, 1 nM sodium selenite, 0.5 mg 1 ml, cultured hygromycin B and geneticin one 1mgml . For the assay, 50,000 cells were inoculated and 1 to 96-well plates and incubated for 24 h at 371C, 5% CO2. The compounds were added to the cells, and.
For 30 min at 371C prior to the addition of increasing concentrations of 50 Nethylcarboxyamideoadenosine After incubation for 3 h at 371C, 5% CO2, 100 ml credited Glo reagent and luminescence was read on a TopCount. A3 receptor binding assay: Chinese hamster ovary cells were stably transfected with the human A3 receptor to confluency in Iscove, s modified Dulbecco’s medium supplemented with 10% f fetal calf serum and 2 mM K Lglutamine bred. For the assay and 1 500 000 cells in 96-well plates were sown t and for 24 h at 371C, 5% CO2. The radioligand, N6-4-amino-3 iodobenzyladenosine methyluronamide 50 N, and increasing concentrations of test compounds were added to the cells and incubated for 120 at 41C.