Whole-cell binding assay

CHO-K1 cells were seeded to 96-well plates (PerkinElmer) coated with fibronectin (Corning) at a density of 30,000 cells per well and incubated overnight. After 24 h transfection, cells were washed twice and incubated with blocking buffer (F12 supplemented with 33 mM HEPES, and 0.1% (w/v) BSA, pH 7.4) for 2 h at 37 °C. Then, radiolabeled ¹²⁵I-GLP-1 (40 pM, PerkinElmer) and increasing concentrations of unlabeled peptide were added and competitively reacted with the cells in binding buffer (PBS supplemented with 10% (w/v) BSA, pH 7.4) at RT for 3 h. After that, cells were washed with ice-cold PBS and lysed by 50 μL lysis buffer (PBS supplemented with 20 mM Tris-HCl and 1% (v/v) Triton X-100, pH 7.4). Finally, 150 μL of scintillation cocktail (OptiPhase SuperMix, PerkinElmer) was employed and radioactivity (counts per minute, CPM) determined by a scintillation counter (MicroBeta2 plate counter, PerkinElmer).