M 24 brandel filtration system

10 μg of membranes from CHO-K1 cells expressing the human 5-HT1A receptor were suspended in 50 mM Tris-HCl, 10 mM MgSO₄, 0.5 mM EDTA, and 0.1% (w/v) ascorbic acid, pH 7.4 and test ligands (91 nM for single point radioligand displacement assay and 1 nM to 1 μM for full competition assay) were incubated with 0.25 nM [³H]8-hydroxy-DPAT (PerkinElmer, Boston, MA, USA) for 60 min at room temperature. The nonspecific binding was determined using 10 μM metergoline (Sigma, St. Louis, MO, USA). After incubation, the bound ligands were filtered using an M-24 Brandel filtration system (Brandel, Gaithersburg, MD, USA), collected on glass fiber papers (Whatman grade 934-AH, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), and the radioactivity was quantitated using a MicroBeta2 Microplate scintillation counter 2450 (PerkinElmer, Boston, MA, USA).

The saturation binding protocols for [³H]DTG and [¹²⁵I]RHM-4 have been previously described [4 (link),6 (link)]. The SD rat liver membranes were incubated with [³H]DTG (0.5–130 nM) for 120 min or with [¹²⁵I]RHM-4 (0.02–9 nM) for 90 min at room temperature. Nonspecific binding in both studies was defined as the presence of 10 μM DTG; 100 nM of (+)-pentazocine was added to the [³H]DTG binding study to mask the sigma-1 binding site. After incubation, the bound ligands from both studies were filtered using an M-24 Brandel filtration system (Brandel, Gaithersburg, MD, USA), collected on glass fiber papers (Whatman grade 934-AH, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), and counted using a MicroBeta2 Microplate counter 2450 or Wizard2 Automatic Gamma Counter 2470. All Kd and Bmax values were calculated via a nonlinear regression method using Prism, and the protein concentrations were determined using Lowry et al.’s method [34 (link)] with BSA as the standard.