Cobra 2 γ counter

Each tube in the binding assay contained 100 μL membrane suspension (20 μg protein), 50 μL [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyl-uronamide (0.2 nM, PerkinElmer, Boston, MA), and 50 μL of increasing concentrations of the test ligands in TrisHCl buffer (50 mM, pH 8.0) containing 10 mM MgCl₂, 1 mM EDTA. Nonspecific binding was determined using 10 μM of 23 in the buffer. The mixtures were incubated at 25 °C for 60 min. Binding reaction was terminated by filtration through Whatman GF/B filters under reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburgh, MD, USA). Filters were washed three times with 9 mL ice-cold buffer. Filters were counted using a PerkinElmer Cobra II γ-counter.

HEK293 cell membrane homogenates (20 μg protein/tube for A₁R, A_2AR, and A₃R; 50 μg protein/tube for A_2BR) or rat brain homogenates (forebrain for A₁R and striatum for A_2AR) were incubated at 25°C for 60 min with various concentrations of test compounds and radioligands ([³H]2 (0.5 nM) for A₁R; [³H]ZM241385 (0.8 nM) for A_2AR); [³H]7 (25 nM) for A_2BR;^{45 (link)–46 (link)} [¹²⁵I]AB-MECA (0.1 nM) for A₃R) in 50 mM Tris.HCl buffer containing 10 mM MgCl₂ Nonspecific binding was determined in the presence of 23 (10 μM for A₁ and A_2A; 100 μM for A_2B and A₃). Binding reaction was terminated by rapid filtration through Whatman GF/B filters using a MT-24 cell harvester (Brandel, Gaithersburg, MD, USA). Filters were washed 3 times with 3 mL of ice-cold buffer. For the binding of [³H]2, [³H]ZM241385, and [³H]7, radioactivity was determined using liquid scintillation counter (Tri-Carb 2810TR; PerkinElmer). For the binding of [¹²⁵I]AB-MECA, radioactivity was analyzed using a PerkinElmer Cobra II γ-counter. Binding parameters were calculated using Prism 7 software (GraphPAD, San Diego, CA, USA). IC₅₀ values were converted to K_i values using the Cheng–Prusoff equation.^{47 (link)} The K_i values from binding experiments are expressed as mean ± standard error from 2–4 independent measurements performed in duplicate.