Isolation and Culture of Mouse Islets

Islets were isolated from 8 to 12 weeks old C57BL/6 male mice as described before by our laboratory [29] (link). Briefly, mouse islets were isolated using perfusion and digestion of pancreas with collagenase V (from Roche), density gradient purification with histoplaque (Sigma), and then hand-picked. Isolated islets were cultured overnight in RPMI 1640 containing 10% FBS, 11 mM glucose, and then switched to RPMI medium containing 3.3 mM glucose for 1 hr before treatment with high concentration of glucose. Insulin levels in the culture media were measured with an ELISA kit from ALPCO. For Ets1 induction of TXNIP studies, 200 isolated mouse islets were also cultured 16 hrs in RPMI 1640 containing 10% FBS and 11 mM glucose and switched to RPMI 1640 with 10% FBS and 20 mM glucose. Ad-EGFP or Ad-Ets1 was then added to islets in culture for 24 hrs at MOI of 100. The islets were collected for Western blot analysis of TXNIP and Ets1.