Glp 1 7 36 nh2

cAMP accumulation was measured using a TR-FRET cAMP kit (PerkinElmer) according to the manufacturer’s instructions. Cells were seeded into 384-well culture plates (1.2 × 10⁴ cells/well) and incubated overnight at 37 °C in 5% CO₂. Upon removal of the culture medium, stimulating solution (5 mL/well containing 0.5 mM IBMX) and 5 μL/well ligand [GLP-1(7–36)NH₂ was purchased from Sigma-Aldrich, and the dual agonists were synthesized by GL Biochem] of different concentrations were added and incubated for 40 min at RT. Five μL Eu-cAMP tracer and 5 μL ULight-anti-cAMP were then introduced followed by 1-h incubation at RT. Signal was detected with an EnVision Multilabel Plate Reader (PerkinElmer). Data were converted using a cAMP standard curve and analyzed using a three-parameter logistic equation.