Mach 3 cell harvester

AT-compounds were dissolved in 100% DMSO to a concentration of 10 mM. The binding assays were performed in 96-well polystyrene plates using six concentrations of each test compound (1 μM–0.01 nM) in triplicate, by adding 100 μL of compound and 100 μL of tritiated ligands [³H]DAMGO (48.0 Ci/mmol, K_d 0.69 nM for MOP) or [³H]N/OFQ (130 Ci/mmol, K_d 0.065 nM for NOP). Nonspecific binding was determined using 1.0 μM of unlabeled N/OFQ for NOP and 1.0 μM of unlabeled DAMGO for MOP. Assays were initiated by the addition of 800 μL of membrane per well, after which the samples were incubated for 60 min at 25 °C in a total volume of 1.0 mL. In NOP receptor experiments, 1 mg/mL BSA was added to the compound dilution buffer. The incubation was terminated by rapid filtration through 0.05% PEI-soaked glass fiber filter mats (GF/C Filtermat A, Perkin-Elmer) on a Tomtec Mach III cell harvester and washed 5 times with 0.5 mL of ice-cold 50 nM Tris–HCl (pH 7.4) buffer. The filters were dried overnight and soaked with scintillation cocktail before counting on a Wallac Beta plate 1205 liquid scintillation counter. Radioactivity was determined as counts per minute (CPM). IC₅₀ values were determined using at least six concentrations of test compound, and calculated using GraphPad/Prism (ISI, San Diego, CA). K_i values were determined by the method of Cheng and Prusoff^{30 (link)}.

The assay is performed in a 96–well polystyrene plate using triplicates of six concentrations of each test compound and tritiated ligands [³H]DAMGO (0.2 nM for MOP), [³H]DPDPE (0.2 nM for DOP), [³H]U69593 (0.2 nM for KOP), or [³H]N/OFQ (0.2 nM for NOP). Nonspecific binding was determined by using 1.0 µM of the unlabeled nociceptin for NOP, 10 µM unlabeled DAMGO for MOP, 10 µM unlabeled DPDPE for DOP, and 10 µM unlabeled U69,593 for KOP. Assays were initiated by addition of membrane homogenates and samples were incubated for 60 min at 25 °C in a total volume of 1.0 mL. In NOP receptor experiments, 1 mg/mL BSA is added to the assay buffer. The amount of protein in the binding assay was 15 μg. The incubation was terminated by rapid filtration through 0.5% PEI-soaked glassfiber filter mats (GF/C Filtermat A, Perkin-Elmer) on a Tomtec Mach III cell harvester and washed 5 times with 0.5 mL of ice-cold 50 nM Tris-HCl, pH 7.4 buffer. The filters were dried overnight and soaked with scintillation cocktail before counting on a Wallac Beta plate 1205 liquid scintillation counter. Radioactivity was determined as counts per minutes (cpm). Full characterization of compounds includes analysis of the data for IC₅₀ values and Hill coefficients using GraphPad Prism. (ISI, San Diego, CA). K_i values were determined by the method of Cheng and Prusoff^{39 (link)}.

PBMCs were isolated from 50 healthy donor buffy coats and CD8+ T cells were depleted using CD8+ RosetteSep (StemCell Technologies). Cells were plated at a density of 4–6 million cells per mL in AIM-V medium (Gibco). On days 5, 6, and 7, cells were gently resuspended in 3 × 100 μL aliquots and transferred to each well of a round bottomed 96 well plate. Cultures were pulsed with 0.75 μCi [³H]-Thymidine (Perkin Elmer) in 100 μL AIM-V culture medium, and incubated for a further 18 h, before harvesting onto filter mats (Perkin Elmer), using a TomTec Mach III cell harvester. Counts per minute (Cpm) for each well were determined by Meltilex (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) in paralux, with low background counting.