Functional analyses of pancreatic islets in glucose homeostasis

 Fast a mouse for 16 h prior to the test.

 Anesthetize a mouse quickly by inhalation of isoflurane to collect 50 μL of blood in a serum separator tube. Measure the blood glucose level using a glucometer.

 Administrate glucose solution at 1.5 g glucose /Kg bodyweight by an intraperitoneal injection using an insulin syringe with a 28-gauge needle (equivalent to an injection of 100 μL of glucose solution for 20 g of body weight), and start a count-up timer.

 Collect blood samples in serum separator tubes at 30, 60, 120, 240 min after glucose injection, and measure blood glucose levels at each time points.

 Following the blood clotting at room temperature for 2 h, separate serum from blood samples by centrifugation at 3,000 × g for 5 min.

 Determine insulin levels in the serum samples using a mouse insulin ELISA kit.

 Euthanize a mouse by inhaling a carbon dioxide and disinfect the mouse with mist of 70% ethanol. Place the mouse on a Styrofoam plate with the abdominal side facing up and incise the upper abdominal skin along the midline followed by holding skin with push pins on the plate and open the peritoneum to expose the liver and intestines.

 Place the mouse under a stereomicroscope, and distally ligate the pancreatic duct. Inject 1mL of collagenase solution into the duct, and then remove the pancreas.

 Transfer the pancreas into 14 mL Falcon tube and add 2 mL of collagenase solution. Incubate pancreas for 14 min at 37°C with shaking (200 strokes/min), and then centrifuge at 200 × g for 5 min.

 Resuspend the resulting cell pellet with 3 mL of 25% Ficoll/HBSS, and then overlay 3 mL of 23% Ficoll/HBSS, 2 mL of 20% Ficoll/HBSS, and 11% Ficoll/HBSS onto the cell suspension to form discontinuous gradients. Centrifuge at 800 × g at room temperature for 15 min, and then collect islet cells from the top two interfaces of gradients using a transfer pipette.

 Dilute cell suspension with HBSS up to 14 mL and centrifuge at 250 × g for 5 min.

 Resuspend islets with 10 mL of HBSS and place in a 10 cm petri-dish.

 Hand pick up islets under a stereomicroscope using a micropipette set to 20 μL, and collect them in a 1.5 mL microtube.

 Consecutively wash islets with 1.5 mL of HBSS, and then incubate them with 1 mL of dispase solution at 37°C for 3 min. Gently and repetitively pipette them until all islets become single cells.

 Wash the single islet cells with 15 mL of HBSS.

 Culture islet cells with PRMI1640 supplemented with 10% FCS, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, and 11 mM glucose.

 Replace the culture medium of islet culture to KRBH containing 2.8 mM glucose (KRBH-2.8G), and incubate at 37°C for 30 min. Take 2×10⁵ islet cells with 2.5 mL syringe and infuse into a 0.2 μm syringe filter.

 Connect the filter with silicon tubes filled with KRBH-2.8G, and set it to a peristaltic pump (flow rate is adjusted to 1 mL/min).

 Equilibrate the cells by infusing KRBH-2.8G sent from a bottle wormed in 37°C water bath for 40 min.

 Start collecting fractions every min. Following the first 10 min infusion with KRBH-2.8G, switch the tube line from KRBH-2.8G to KRBH-16.8G and continue to collect fractions following 20 min.

 Determine the insulin levels in fractions by Insulin ELISA kit.

 Harvest cultured islet cells with ice-cold 2 mM EDTA in PBS and wash with ice-cold FACS buffer.

 Incubate 5×10⁴ islet cells with Glut2 antibody (1:100 dilution) in 100 μL of FACS buffer on ice for 15 min, and wash cells with 200 μL of ice-cold FACS buffer 3 times.

 Incubate cells with FITC-conjugated sheep anti-rabbit IgG (1:200 dilution) in 100 μL of FACS buffer on ice for 15 min, and wash cells with 200 μL of ice-cold FACS buffer 3 times.

 Resuspend cells with 300 μL of FACS buffer, and subject to flow cytometric analysis using FACSCalibur Flow Cytometer with CellQuest software.