Isoplate 96

Crude membranes (5 μg/ml) were incubated in various concentrations of cold paroxetine (0.003–100 nM depending on SERT mutant) in Ringer’s for 1 hr. Binding was initiated by adding [¹²⁵I]RTI-55 (β-carbomethoxy-3β-[4-iodophenyl]tropane); specific activity 2,200 Ci/mmol, Perkin Elmer, NEX272) to the membrane-paroxetine mix at a final concentration of 0.2 nM and then incubating with gyration in glass tubes for 1 hr at RT. Nonspecific binding was determined by incubating the identical mixes in the presence of 200 μM cold paroxetine. To separate bound from free [¹²⁵I]RTI-55, reactions were vacuum-filtered through a 96-well Multiscreen FB filter plate (MSFBN6B, Millipore) that had been pre-treated with 0.6% polyethyleneimine for 4–16 hrs, and then washed with 6 × 200-μl aliquots of Ringer’s. Filters were transferred to a 96-well Isoplate-96 (Perkin Elmer), scintillation fluid added, and counted in a Packard MicroBeta scintillation counter. To avoid ligand depletion, total binding counts was always less than 10% counts added. Experiments were performed at least 3 times, each time in triplicate, and data fit to a sigmoidal dose-response curve, as implemented in GraphPad Prism 6. Student’s unpaired t-test was used for statistical comparison.

Freshly isolated and purified mouse NK cells were tested for their capacity to trigger NK cytotoxicity in redirected cytolysis assays with antibody-loaded FcγR⁺ P815 cells as target cells. NK cells were preactivated in vivo by poly(I:C) injection 16 h before harvest. P815 cells were labeled with 50 μCi of ⁵¹Cr (Perkin Elmer, Waltham, MA, USA) for 2 h at 37°C. After washing, ⁵¹Cr-labeled P815 cells (targets) were co–cultured for 4 h with purified NK cells (effectors) in the presence of 1 µg/ml of the indicated antibodies at different effector (E) to target (T) ratios. Subsequently, supernatants were mixed with OptiPhase Supermix scintillation mixture in an IsoPlate-96 and measured with a MicroBeta2 plate counter (all from PerkinElmer). Spontaneous chromium release of target cells was always less than 15% of the maximum release of target cells lysed in 1% Triton X-100. Percentage of lysis was calculated as follows: 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release). Experiments were performed in triplicates.