Pancreatic islet perifusion.

For islet perifusion, 75 islets were handpicked and loaded into 0.275 mL chambers containing KRPH (140 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl₂, 1 mM NaH₂PO₄, 1 mM MgSO₄, 5 mM HEPES, 2 mM NaHCO₃, 1% fatty acid-free BSA) in 2.7 mM glucose. Upon initiation of the perifusion experiment, KRPH with 2.7 mM glucose + 1% BSA was perifused at a rate of 200 μL/min for 24 minutes, followed by KRPH with 2.7 mM glucose +0.1% BSA, to provide an equilibration period for the islets. Following equilibration, experimental conditions were applied, and perifusate was collected each minute. GLP-1 and GIP were diluted in KRPH prior to experiment. Perifusate insulin concentrations were measured with AlphaLISA (PerkinElmer). The following concentrations were used for each reagent: GLP-1, 300 pM; GIP, 3 nM; tirzepatide, 1 nM; GIPR antagonist, 300 nM; exendin-4_(9–39) (Ex9), 1 μM.