P e 1450 microbeta wallac trilux scintillation counter

Membranes from HEK293 cells overexpressing hERG were prepared and stored as described previously (Yu et al, 2014). Single‐point dissociation assay was performed in incubation buffer (10 mM HEPES, 130 mM NaCl, 60 mM KCl, 0.8 mM MgCl₂, 1 mM EGTA, 10 mM glucose, 0.1% BSA, pH 7.4) as detailed previously (Yu et al, 2015). Briefly, the dissociation of [³H]dofetilide was initiated by addition of 10 μM dofetilide in the absence (control) or presence of 10 or 50 μM LUF compounds after pre‐incubation at 25°C for 2 h. After 6 min of dissociation, incubations were terminated by dilution with ice‐cold wash buffer (25 mM Tris–HCl, 130 mM NaCl, 60 mM KCl, 0.8 mM MgCl₂, 0.05 mM CaCl₂, 0.05% BSA, pH 7.4). Separation of bound from free radioligand was performed by rapid filtration through a 96‐well GF/B filter plate using a PerkinElmer Filtermate‐harvester PerkinElmer (MA, USA). The filter‐bound radioactivity was determined by scintillation spectrometry using the P‐E 1450 Microbeta Wallac Trilux scintillation counter (PerkinElmer, MA, USA) after addition of 25 μl Microscint and 2‐h extraction. Data were analysed with GraphPad Prism 6.0 or 7.0 (GraphPad Software, CA, USA) and R 3.2.4 (The R Foundation for Statistical Computing).

Radioligand displacement experiments were performed as follows. Membrane aliquots containing 10 μg of protein were incubated in a total volume of 100 μL of assay buffer to adjust the assay window to approximately 3000 dpm. Nonspecific binding was determined in the presence of 100 μM NECA and represented less than 10% of the total binding. Then, to each tube were added 25 μL cell membrane (10 μg of protein), 25 μL of 2.7 nM radioligand [³H] ZM241383, 25 μL of assay buffer [25 mM Tris-HCl, pH 7.4 at 25 °C , supplemented with 5 mM MgCl₂ and 0.1% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS)], and 25 μL of the indicated compounds in increasing concentrations in the same assay buffer. The mixture was incubated at 25 °C for 60 min to reach equilibrium. Incubations were terminated by rapid vacuum filtration to separate the bound and free radioligand through 96-well GF/B filter plates using a Perkin Elmer Filtermate-harvester (PerkinElmer, Groningen, Netherlands). Filters were subsequently washed three times with 2 mL of ice-cold buffer (25 mM Tris-HCl, pH 7.4, supplemented with 5 mM MgCl₂). The filter-bound radioactivity was determined by scintillation spectrometry using a P-E 1450 Microbeta Wallac Trilux scintillation counter (PerkinElmer).