Islet Perifusion Protocol for Insulin Secretion

Isolated islets (from the same groups as above) recovered overnight in CMRL-1066 medium (Gibco) containing 10% fetal bovine serum and 2 mmol/l L-glutamine at 37°C. Thirty islets per NHP were placed in a dynamic perifusion system (Amersham Biosciences AKTA FPLC System) as previously described^{33 (link)}. To summarize, the perifusion was performed using Krebs buffer with 2.8 mM glucose at a flow rate of 1 ml/min for 30 minutes to establish stable basal insulin secretion. Next, the islets were perifused with 2.8 mM glucose for 10 minutes, and fractions of 500 μl were collected every 30 seconds. Then, the glucose concentration was increased to 20 mM, and fractions of 500 μl were collected every 30 seconds for 20 minutes. Finally, the islets were perifused with 30 mM KCl, and fractions of 500 μl were collected every 30 seconds for 10 minutes. After the perifusion, the islets were recollected from the column for protein quantification. The insulin in the effluent was measured as described above. The fractional insulin secretion rate was calculated as secreted insulin per minute normalized to the protein content.