Optiphase supermix

A total of 1 × 10⁶ target cells were dissociated from plates, washed twice in RPMI, resuspended in 100 µl of a 7.4 MBq ${\text{Na}}_2^{51}{\text{CrO}}_4$ solution (GE Healthcare Life Sciences, Piscataway, NJ, USA), diluted in growth media, and then incubated for 1 h in a humidified 5% CO₂ atmosphere at 37°C. After washing three times with RPMI, the labeled cells were resuspended in their growth media and seeded in 96-well plates at 5 × 10⁴ cells per well in quadruplicate. Effector cells were added at the specified effector to target cell ratios (E:T) and were incubated for 4 h at 37°C, 5% CO₂. The supernatant was removed, added to Optiphase Supermix (PerkinElmer), and radioactivity measured with a beta counter (MicroBeta TriLux, PerkinElmer, Waltham, MA, USA). The degree of cytotoxicity was determined according to the formula: % cytotoxicity = [(sample release) − (spontaneous release)]/[(maximum release) − (spontaneous release)]. Spontaneous release was determined by incubating target cells in medium alone. Maximum release of target cells was measured following treatment with 10% detergent Triton X-100 (Sigma). Error bars represent the SD error of the acquired values. To test for significance, a Student’s t-test was performed with the degree of cytotoxicity values of each ratio against the other conditions. *p < 0.05, **p < 0.01.

Cells were seeded on 24-well plates at a density of 1 × 10⁵ cells/well for 2 days before experiment. Cells were subsequently incubated with Na⁺ containing buffer (125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 25 mM HEPES, 1.2 mM KH₂PO₄ and 5.6 mM glucose, pH 7.4) or Na⁺-free buffer (125 mM choline chloride, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 25 mM HEPES, 1.2 mM KH₂PO₄ and 5.6 mM glucose, pH 7.4) containing 19 µM leucine, 1 µM [¹⁴C] l-leucine (> 8.14 GBq/mmol, Moravek, CA, USA), and 0.01–100 μM JPH203 for 2 min at 37 °C. The cells were solubilized with 0.1 N NaOH and cell lysates were added to OptiPhase SuperMix (PerkinElmer, Waltham, MA, USA). The radioactivity was measured by a liquid scintillation counter; LSC-7400 (Hitachi-Aloka Medical, Tokyo, Japan).

The ability of VxXIIB variants to displace the binding of [³H]-epibatidine to the recombinantly expressed Ls-AChBP was determined in competitive radioligand binding assays. Briefly, [³H]-epibatidine (1 nM final concentration) and increasing concentrations of test ligand in a final volume of 100 ml were incubated in 96-well plates (Flexible PET Microplate, Perkin Elmer) precoated with 1 ng/ml of Ls-AChBP per well in binding buffer (phosphate buffered saline with 0.05% bovine serum albumin). The mixture was then removed and 100 ml of scintillant (Optiphase Supermix, Perkin Elmer) added to each well. Bound radioactivity was measured with a Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer).

Cardiomyocytes cultured in 12-well plates were incubated in glucose-free medium and treated with 100 nM insulin (Humalog, Eli Lilly and Company, Indianapolis, IN) for 30 minutes. To measure glucose uptake, 0.2 mM 2-deoxyglucose mixed with 0.077 µCi/ml [14C]2-deoxyglucose (Perkin-Elmer, Waltham, MA) was added to the cell culture medium for additional 30 minutes (specific activity 385 µCi/mmol). The cells were washed three times in PBS, harvested in 0.1 M NaOH and the lysed cells counted by conventional liquid scintillation with OptiPhase SuperMix scintillation fluid (Perkin-Elmer). Glucose uptake was normalized to the cell lysate protein content.

100,000 (24-h assay) or 200,000 (15-min assay) U2OS or ARN8 cells were seeded per well in 6-well plates in 2 ml of fully supplemented culture medium as described above. 24 h postseeding, medium was removed, and cells were treated with the indicated compounds for either 15 min or 24 h in 2 ml of FBS-free medium supplemented with serum replacement 3 (Sigma–Aldrich #S2640). At the end of the treatment, an extra well of each treatment was harvested in 100 μl of 1× LDS (62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 1% LDS) for quantification of protein levels to determine the level of cell death after 24 h for normalization with protein concentration determined by the Bio-Rad DC protein assay (Bio-Rad #5000111). [³H]Uridine (PerkinElmer Life Sciences #NET367001MC) was prepared in serum replacement medium to a final emission of 3.65 μCi ml⁻¹ and added to cells for 10 min. Following 10 min, the cells were washed with four quick changes of 1 mm unlabeled uridine prepared in ice-cold transport buffer (20 mm Tris-HCl, pH 7.4, 130 mm NaCl, 3 mm K₂HPO₄, 1 mm MgCl₂, 5 mm glucose, 2 mm CaCl₂). The samples were then harvested in 100 μl of 10% SDS. 900 μl of Optiphase Supermix (PerkinElmer Life Sciences #1200-439) was added to each sample, and the cpm were measured using a 1450 MicroBeta JET for liquid scintillation (PerkinElmer Life Sciences/Wallac).

Competitive radioligand binding assay with ³H-epibatidine (specific activity 1.11–2.59 TBq/mmol) and nAChR agonists and antagonists was used to determine the functional activity of the recombinant Ls- and Ac-AChBPs. The proteins were coated onto 96 well plates (Flexible PET Microplate, Perkin Elmer) at a concentration of 8 μM total protein. Serial dilutions of nAChR ligands together with a fixed concentration of ³H-epibatidine (1 nM) were incubated with the immobilized protein in 100 μL of assay buffer (phosphate buffered saline with 0.05% bovine serum albumin) for 1 h at 4°C. Unbound ligands were washed manually followed by addition of 100 μL scintillant (Optiphase Supermix, Perkin Elmer). Plates were incubated with the scintillant for 2 min on a shaker and radioactivity measured with a Wallac 1450 MicroBeta liquid scintillation counter. Binding data were evaluated by a nonlinear, least squares one-site competition fitting procedure using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). The radioligand binding assays were performed in triplicate in three separate experiments (n = 3), with IC₅₀ values reported as mean ± standard error of the mean (S.E.M).