Adenosine A2A Receptor Radioligand Binding Assay

FreeStyle HEK293-F cells transiently expressing wild type A_2AR were resuspended in either assay buffer A (25 mM HEPES, pH 7.5, 100 mM KCl, 1 mM MgCl₂), assay buffer B (25 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl₂), or assay buffer C (25 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM MgCl₂), and were lysed by 10 passages through a 26-gauge needle. Purified binding partners were buffer-exchanged to the respective buffer before being added to the membranes at a final concentration of 25 μM. The mixture was aliquoted and NECA was added (0 to 1 mM final concentration, prepared in assay buffers containing 1 u/mL apyrase). The samples were incubated for 90 min at 22°C, ³H-ZM241385 was added at its apparent K_d (2.5 nM) and allowed to bind for a further 90 min at 22°C. Non-specific binding was determined in the presence of 100 μM of ZM241385. Receptor-bound and free radioligand were separated by filtration through 96-well GF/B filter plates (pre-soaked with 0.1% polyethyleneimine), and washed 3 times with the appropriate buffer. Plates were dried and radioactivity was quantified by liquid scintillation counting using a Tri-Carb 2910 TR (Perkin Elmer). Data were analyzed by nonlinear regression using GraphPad Prism software. The K_i for NECA binding was derived from one-site fit K_i analysis. Data from at least three independent experiments, each performed in duplicate, were analyzed using an unpaired two-tailed t-test for statistical significance.