Microscint 0 scintillant cocktail

Transfected HEK293T cells were harvested and resuspended in 50 mM cold TrisHCl buffer (pH 7.4). Cell membrane preparations were made by centrifugation at 33,000 × g for 20 min at 4 °C. Membrane pellets were homogenized for 30 s using the T-25 Ultra-Turrax homogenizer (Ika). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) and normalized across samples prior to the assay. Binding assays were performed in 96-well plates, where samples were incubated with 10 nM (for α7) [³H]epibatidine for 3 h at room temperature. Nonspecific binding was determined by coincubation with 10 μM unlabeled epibatidine. Binding assays were terminated by filtration through polyethylenimine-treated Unifilter GF/B 96-well plates (PerkinElmer), which were immediately washed with 500 mL assay buffer per plate. Filter plates were desiccated at 65 °C for 20 min and incubated with 50 μL MicroScint-0 scintillant cocktail (PerkinElmer). Bound [³H]epibatidine was quantified using the TopCount NXT scintillation counter (PerkinElmer).

HEK293T cells or cortical neurons were harvested in 50 mM ice-cold TrisHCl buffer (pH 7.4). Cells were homogenized for 30 s using the T-25 Ultra-Turrax homogenizer (Ika) and total protein concentration of the homogenate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Cell homogenates were incubated with 10 nM [³H]epibatidine (for nAChR) or with 30 nM [³H]flunitrazepam (for GABA) in 96-well plates for 3 h at room temperature. Nonspecific binding was determined by co-incubation of the cell samples with 10 μM unlabeled epibatidine or 100 μM unlabeled flunitrazepam. Assays were terminated by filtration through polyethylenimine-treated 96-well Unifilter GF/B plates (PerkinElmer). Filter plates were washed with 500 mL TrisHCL buffer and then desiccated at 65 °C for 30 min. MicroScint-0 scintillant cocktail (50 μL; PerkinElmer) was added to each well and plates read with a TopCount NXT scintillation counter (PerkinElmer).