Heated rodent operating table

Briefly, the aorta was ligated above the coeliac artery and below the superior mesenteric artery and then cannulated. The pancreas was perfused at 1.34 µl min⁻¹ mg⁻¹ pancreas weight using an Ismatec Reglo Digital MS2/12 peristaltic pump. Pancreatic weight was estimated from whole body weight as previously described [18 (link), 19 (link)]. The perfusate was maintained at 37°C using a Warner Instruments temperature control unit TC-32 4B in conjunction with an in-line heater (Warner Instruments P/N 64-0102) and a Harvard Apparatus heated rodent operating table. The effluent was collected in intervals of 1 min into 96-well plates kept on ice and containing aprotinin. Samples were subsequently stored at −80°C pending analysis of glucagon content (using the Mercodia assay).

Briefly, the aorta was ligated above the coeliac artery and below the superior mesenteric artery and then cannulated. The pancreas was perfused with KRB containing varying concentrations of glucose and somatostatin-14 (Tocris, Cat. No 1157) as indicated in the figures, at a speed of 1.34 μL/min/mg pancreas weight using an Ismatec REGLO Digital MS2/12 peristaltic pump. Pancreatic weight was estimated from the whole body weight as previously described [33 ,34 ]. The perfusate was maintained at 37 °C using a Warner Instruments temperature control unit TC-32 4B in conjunction with an in-line heater (Warner Instruments P/N 64-0102) and a Harvard Apparatus heated rodent operating table. The effluent was collected in intervals of 1 min into 96-well plates which were kept on ice and contained aprotinin. Samples were subsequently stored at −80 °C pending analysis of glucagon content.

Dynamic measurements of glucagon were performed using the in situ perfused mouse pancreas. Briefly, the aorta was cannulated by ligating above the coeliac artery and below the superior mesenteric artery, and the pancreas was perfused with KRB at a rate of ~0.45 ml/min using an Ismatec Reglo Digital MS2/12 peristaltic pump. The KRB solution was maintained at 37°C with a Warner Instruments temperature control unit TC-32 4B in conjunction with a tube heater (Warner Instruments P/N 64-0102) and a Harvard Apparatus heated rodent operating table. The effluent was collected by cannulating the portal vein and using a Teledyne ISCO Foxy R1 fraction collector. The pancreas was first perfused for 10 min with 3 mM glucose before commencing the experiment to establish the basal rate of secretion. Glucagon measurements in collected effluent were performed using RIA.