Assay Method Information
Assay Name:  Characterization of Agonism at Human D3 Receptors Using Cyclic Adenosine Monophosphate Detection
Description:  Agonist activity at human D3 receptors was assayed by cAMP levels in HEK293 cells expressing recombinant human D3 receptors (BioXtal, Saint-Félix, France) stably co-expressing adenylyl cyclase V (ACV) (cell line developed by Gedeon Richter) by homogenous time-resolved fluorescence (HTRF) using the cAMP Gi kit (Cisbio/PerkinElmer). HEK293 cells expressing recombinant human D3 receptors and ACV were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin antimycotic solution, 1% pyruvate, 100 μg/mL G418 (Thermo Fisher Scientific) and 60 μg/mL hygromycin B and maintained at 37° C. in a humidified atmosphere containing 5% CO2. Prior to measuring cAMP, cells were detached with Versene (Thermo-Fisher Scientific) and resuspended in assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 10 mM glucose, pH 7.4) in white-walled, half area 96-well microplates at a density of 15,000 cells/well and 20 μL volume. The assay buffer was supplemented with 100 μM IBMX (Sigma Aldrich, St Louis, MO, USA). After adding the test compounds (4×concentrated) or vehicle (DMSO) at 10 μL/well, cells were incubated with assay buffer or various concentrations of the test compounds for 20 minutes. After an additional 30 minutes incubation at ambient temperature with 1.5 μM forskolin (DMSO final concentration was 0.3%) cell stimulation was stopped by adding detection reagents (20 μL cAMP-d2 and 20 μL anti-cAMP cryptate) diluted in lysis buffer (PerkinElmer). The time-resolved fluorescence (TRF) signal was quantified with a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) using standard HTRF settings with laser excitation at 337 nm after 60 minutes of incubation at ambient temperature.
Affinity data for this assay
 
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