Pancreatic islets were isolated from GLP-1R-iCre-GCaMP3 mice (9–13 weeks old), a mouse strain in which GCaMP3 is restricted to cells with GLP-1R promoter activity [22 (link)]. Pancreata were inflated with RPMI-1640 medium containing 1 mg/mL collagenase from Clostridium histolyticum (S1745602, Nordmark Biochemicals, Germany), dissected and incubated in a water-bath at 37 °C for 12 min. Islets were subsequently washed and purified using a Histopaque gradient (Histopaque-1119 and -1083). Isolated islets were allowed to recover overnight in RPMI-1640 supplemented with 10% and 1% penicillin/streptomycin. Islet dispersal was achieved by trituration in 0.05% trypsin-EDTA at 37 °C for 3 min, followed by trypsin inactivation with complete medium. Dispersed islet cells were then seeded onto poly-D-lysine-coated coverslips and allowed to adhere overnight in complete RPMI-1640. The following day, 100 nM GLP-1R-TMR, exendin-4-TMR, or vehicle were added to the well, and incubated for 30 min at 37 °C. Medium was then removed, and cells were washed once with HBSS and fixed with 4% PFA for 15 min. Washed coverslips were subsequently mounted onto glass slides in Prolong Diamond anti-fade with DAPI (Thermo Fisher).
Free full text: Click here
