Ultima gold xr scintillation fluid

EIDD-1931, EIDD2801, and Nirmatrelvir (PF-073213332) were purchased from MedChemExpress (Shanghai, China). Stock solutions of the compounds were freshly prepared in dimethyl sulfoxide (DMSO). Non-radiolabeled chemicals were obtained from Merck/Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of analytical grade. ³H-Uridine ([5-³H], 20.9 Ci/mmol), and ³H-Adenosine ([2,8-³H], 29.3 Ci/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA, USA). Ultima Gold XR scintillation fluid was purchased from PerkinElmer (Waltham, MA, USA).

Membrane proteins prepared from Sf9 cells expressing dopamine D₂ (25 μg) or dopamine D₃ receptors (10 μg) were incubated with a range of concentrations of [³H]spiperone in buffer 2 (20 mM HEPES, 6 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, pH 7.4) supplemented, where appropriate, with 100 mM NaCl or 100 mM N-methyl-D-glucamine (NMDG). Reactions were performed, in triplicate, in 4 ml LP4 test tubes (1 ml final volume) and were initiated by addition of membrane proteins. Non-specific binding was determined in the presence of (+)-butaclamol (3 μM). Reactions were incubated for 3 hours at 25°C and were terminated by rapid filtration through Whatman glass microfibre GF/C filters using a Brandel cell harvester. After four 3 ml washes with PBS (4°C) filter discs were transferred to scintillation vials and soaked in 2ml Ultima Gold^™ XR scintillation fluid (Perkin Elmer) for at least 6 hours prior to their radioactivity being determined by liquid scintillation spectrometry. Specific binding was calculated by subtraction of non-specific binding and free radioligand concentration, corrected for ligand depletion calculated. Data were analysed using Prism (Graphpad) and were fitted to hyperbolic equations describing a one-binding site model.

³H‐prazosin, ³H‐adenine, Microscint 20, Ultima Gold XR scintillation fluid and the Surefire Alphascreen pERK1/2 kit were from PerkinElmer. ¹⁴C‐cAMP was from Hartmann Analytic. Fluo‐4AM and pluronic F‐127 were from Invitrogen. Gibco foetal bovine serum was from Fischer Scientific. All other reagents were from Sigma‐Aldrich. A list of the ligands studied with the source and supplier code is given in Table S1.