Charcoal dextran

Islets of Langerhans were isolated from two mice in each group by using the collagenase digestion method, as previously described (24 (link)). Islets were then cultured for 20–24 h in RPMI-1640 medium supplemented with 11 mM glucose (Invitrogen, CA, USA), 2 mM glutamine, 200 IU/ml penicillin, 200 μg/ml streptomycin and 8% fetal bovine serum stripped with charcoal-dextran (Invitrogen). For GSIS experiments islets were pre-incubated at 37 °C for 2 h in Krebs-bicarbonate buffer solution containing 14 mM NaCl, 0.45 mM KCl, 0.25 mM CaCl₂, 0.1 mM MgCl₂, 2 mM HEPES and 3 mM glucose, and equilibrated with 95% O₂: 5% CO₂ at pH 7.4. Size-matched islets, five in each well from a 24-well plate, were seeded in 0.5 mL fresh buffer containing 3 mM glucose or 11 mM glucose. Then islets were incubated for 1 h at 37°C, 5% CO₂. After incubation, 1% bovine albumin was added to each well, and the plate was cooled at 4°C for 15 min to stop insulin secretion. Media were then collected and stored at −20°C until insulin measurement by ELISA (Mercodia, Uppsala, Sweden), according to the manufacturer's instructions.

Mouse islets of Langerhans were isolated by using the collagenase digestion method, as previously described^{40 (link)}. Islets were then cultured for 20–24 hours in RPMI-1640 medium supplemented with 11 mM glucose (Invitrogen, CA, USA), 2 mM glutamine, 200 IU/ml penicillin, 200 µg/ml streptomycin and 8% fetal bovine serum stripped with charcoal-dextran (Invitrogen). GSIS experiments were performed as previously described^{40 (link)}. Briefly, islets were first pre-incubated at 37 °C for 2 h in Krebs-bicarbonate buffer solution containing (in mM): 14 NaCl, 0.45 KCl, 0.25 CaCl2, 0.1 MgCl2, 2 HEPES and 3 glucose, and equilibrated with 95% O2: 5% CO2 at pH 7.4. Size-matched islets, five in each well, were seeded in 0.5 mL fresh buffer containing 3 mM glucose or 11 mM glucose and further incubated for 1 h. After incubation, 1% bovine albumin was added to each well, and the plate was cooled at 4 °C for 15 minutes to stop insulin secretion. The media were then collected and stored at −20 °C until insulin measurement by ELISA (Mercodia, Uppsala, Sweden), according to the manufacturer’s instructions.