Radioligand binding assays at human and rat A2A adenosine receptors

Competition binding experiments using the agonist radioligand [³H]{"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053"}}CGS21680 were performed in a final volume of 400 µL containing 10 µL of test compound dissolved in DMSO, 190 µL buffer (50 mM Tris–HCl, 10 mM MgCl₂, pH 7.4), 100 µL of radioligand solution in the same buffer (final concentration 5 nM), and 100 µL of membrane preparation (human: 4 µg of protein per vial in buffer, recombinantly expressed in CHO cells; rat: rat brain striatal membrane preparation, 200 µg protein per vial). Non-specific binding was determined in the presence of 5′-N-ethylcarboxamidoadenosine (NECA; final concentration 10 µM) and the incubation period was 120 min at room temperature.