Gf c multiscreen plate

Adenosine A_2A receptor competition binding experiments were carried out in membranes from HeLa-A_2A cells. On the day of assay, membranes were defrosted and re-suspended in incubation buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl₂ and 2 UI/mL adenosine deaminase (pH = 7.4). Each reaction well of a GF/C multiscreen plate (Millipore, Madrid, Spain), prepared in duplicate, contained 10 μg of protein, 3 nM [³H]ZM241385 and test compound. Nonspecific binding was determined in the presence of 50 μM NECA. The reaction mixture was incubated at 25 °C for 30 min, after which samples were filtered and measured in a microplate beta scintillation counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain).

Adenosine A₁ receptor competition binding experiments were carried out in membranes from CHO-A₁ cells (Euroscreen, Gosselies, Belgium). On the day of assay, membranes were defrosted and re-suspended in incubation buffer 20 mM Hepes, 100 mM NaCl, 10 mM MgCl₂, 2 UI/mL adenosine deaminase (pH = 7.4). Each reaction well of a GF/C multiscreen plate (Millipore, Madrid, Spain), prepared in duplicate, contained 15 μg of protein, 2 nM [³H]DPCPX and test compound. Nonspecific binding was determined in the presence of 10 μM (R)-PIA. The reaction mixture was incubated at 25 °C for 60 min, after which samples were filtered and measured in a microplate beta scintillation counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain).