Multiscreen vacuum manifold 96 well

[³H]-raclopride binding was displaced by quinpirole to determine the proportion of receptors in the high-affinity state (RH), the high-affinity (Ki, High), and low-affinity (Ki, Low) values. Nucleus accumbens membrane preparations (60 μg protein/mL) were incubated with increasing concentrations of quinpirole (0.01 nM to 1 mM) and 2 nM [³H]-raclopride (75 Ci/mmol, Novandi Chemistry AB, Sweden) in 250 μL of incubation buffer (50 mM Tris-HCl, 100 mM NaCl, 7 mM MgCl2, 1 mM EDTA, 0.05% BSA, 1 mM DTT) and 0.3 IU/mL adenosine deaminase (EC 3.5.4.4, Sigma-Aldrich) for 90 min at 30 °C in the presence or absence of 100 nM of the A2AR agonist CGS 21680. Nonspecific binding was defined by radioligand binding in the presence of 100 μM (+)-butaclamol (Sigma-Aldrich, Sweden). The incubation was terminated by rapid filtration Whatman GF/B filters (Millipore Corp, Sweden) using a MultiScreenTM Vacuum Manifold 96-well followed by five washes (250 μL per wash) with ice-cold washing buffer (50 mM Tris-HCl pH 7.4). The filters were dried, 5 mL of scintillation cocktail was added, and the amount of bound ligand was determined after 12 h by liquid scintillation spectrometry.

[³H]-raclopride binding was displaced by quinpirole to determine the proportion of receptors in the high-affinity state (RH), the high-affinity value (Ki, High), and the low-affinity (Ki, Low) value. Ventral striatum membrane preparations (60 μg protein/ml) were incubated with increasing concentrations of quinpirole (0.01 nM to 1 mM) and 2 nM [³H]-raclopride (75 Ci/mmol, Novandi Chemistry AB, Sweden) in 250 μl of incubation buffer (50 mM Tris-HCl, 100 mM NaCl, 7 mM MgCl₂, 1 mM EDTA, 0.05% BSA, 1 mM DTT) and 0.3 IU/ml adenosine deaminase (EC 3.5.4.4, Sigma-Aldrich). The incubation took place for 90 min at 30°C in the presence or absence of 100 nM of the A2AR agonist CGS-21680. Non-specific binding was defined by radioligand binding in the presence of 100 μM (+)-butaclamol (Sigma-Aldrich, Sweden). The incubation was terminated by rapid filtration using Whatman GF/B filters (Millipore Corp, Sweden) and a MultiScreenTM Vacuum Manifold 96-well, followed by five washes (250 μl per wash) with ice-cold washing buffer (50 mM Tris-HCl pH 7.4). The filters were dried, 5 ml of scintillation cocktail was added, and the amount of bound ligand was determined after 12 h by liquid scintillation spectrometry.

[³H]-Raclopride binding was displaced by quinpirole to determine the proportion of receptors in the high affinity state (RH), the high affinity (K_i,High), and low affinity (K_i,Low) values for the agonist binding sites from competition curves in HEK cells expressing either A_2AR/3xHA-D₂R, A_2AR/3xHA-D₂R(Tyr192Ala^5.41x42), or A_2AR/3xHA-D₂R(Leu207Ala^5.56/Lys211Ala^5.60). Membrane preparations (60 μg protein/ml) were incubated with increasing concentrations of quinpirole (0.001 nM to 1 μM) and 2 nM [³H]-raclopride (75 Ci/mmol, Novandi Chemistry AB, Sweden) in 250 μl of incubation buffer (50 mM Tris-HCl, 100 mM NaCl, 7 mM MgCl₂, 1 mM EDTA, 0.05% BSA, 1 mM DTT) and 0.3 IU/ml adenosine deaminase (EC 3.5.4.4, Sigma-Aldrich) for 90 min at 30°C in the presence or absence of 100 nM of the A_2AR agonist CGS-21680. Non-specific binding was defined by radioligand binding in the presence of 10 μM (+) butaclamol (Sigma-Aldrich, Sweden). The incubation was terminated by rapid filtration Whatman GF/B filters (Millipore Corp, Sweden) using a MultiScreen^TM Vacuum Manifold 96-well followed by three washes (∼250 μl per wash) with ice-cold washing buffer (50 mM Tris-HCl pH 7.4). The filters were dried, 5 ml of scintillation cocktail was added, and the amount of bound ligand was determined after 12 h by liquid scintillation spectrometry.