Insulin secretion in isolated murine pancreatic islets

Pancreatic islets were isolated from mice by collagenase digestion and Dextran density gradient separation and were cultured overnight in RPMI-1640 medium containing 11 mM glucose, 10% FBS, and 2 mM glutamine. After overnight recovery, islets were incubated in KRB buffer with 2.8 mM glucose and 0.5% BSA for 45 min, transferred to a 48-well plate with 4 islets per well containing 300 μl/well of compound solutions prepared in appropriate glucose concentrations and 0.1% BSA, and incubated at 37°C for 60 minutes. Secretion was stopped by refrigerating the plates at 4°C for 3 minutes. Supernatant was removed from the wells and assayed for insulin levels using the MA6000 Mouse/Rat Insulin Kit (Meso Scale Discovery).