Wallac gamma counter

Blood samples were collected in EDTA–containing tubes, centrifuged, placed in aliquot tubes and stored at −70 to −80°C until post trial batch (with samples from each participant run in the same assay) analysis of metabolites (insulin, glucose, free fatty acids [FFA], and triglycerides [TG]) and appetite–related hormones (total ghrelin, total PYY, total GLP‐1, and leptin). With the exception of leptin, which was only measured in the fasting state, the area under the curve (AUC) for all laboratory measures was calculated using the trapezoid method (Allison et al. 1995). For GLP‐1, 30 μL of dipeptidyl peptidase IV inhibitor was added to the 4 mL EDTA tube prior to collection. GLP‐1 assays were performed with Alpco Diagnostics ELISA (43‐GPTHU‐E01). Insulin concentrations were measured using competitive radioimmunoassay (Millipore). Radioimmunoassay was used to analyze serum leptin (Millipore), serum PYY concentrations (Millipore Cat. #PYYT‐66HK) and total serum ghrelin concentrations (Millipore Cat. #GHRT‐89HK). All radioimmunoassays were performed with a Perkin Elmer Wallac Gamma counter using Maciel RIA‐AID data reduction software. Assays for glucose, TG and FFA were performed on the Olympus AU400e Chemistry Analyzer (Beckman). Reagents were purchased from Beckman Coulter for glucose and TG and from WACO for FFA.

One day before transfection, the COS-7 cells were seeded in 75 cm² culture flasks (3 million cells/flask) in DMEM, containing 3.9 g/L NaHCO₃ and supplemented with 10% FBS, 1% l-glutamine, 180 units/mL penicillin and 45 µg/mL streptomycin. After an overnight culture at 37 °C, 10% CO₂ and 95% air humidity, the cells were transiently transfected with different GLP-2R constructs or pcDNA3.1 vector as previously described.^{52 (link)} Following 24-h incubation, the cells were transferred to Costa® microtiter plates (150,000 cells/well, PerkinElmer) to achieve a 5%–10% specific binding. Forty-eight hours after transfection, the cells were washed twice in binding buffer (50 mM HEPES, pH 7.4, supplemented with 1 mM CaCl₂, 5 mM MgCl₂ and 0.5% (w/v) BSA) and incubated for 15 min at 4 °C. An increasing concentration of unlabeled GLP-2(1–33) or teduglutide (ranging from 0.1 nM to 1 μM) followed by a fixed concentration of [¹²⁵I]GLP-2(1–33, M10Y) (10–15 pM) was added to the cells and incubated for 4 h at 4 °C. The cells were then washed twice in binding buffer (4 °C), lysed and counted for radioactivity using a Wallac Gamma Counter (PerkinElmer).