Murine Pancreatic Islet Isolation

Mice were maintained according to the guidelines of the Canadian Council on Animal Care. All protocols were approved by the University of British Columbia Animal Care Committee. Islets were isolated as previously described [30 (link),69 (link)]. Briefly, pancreata from both male and female mice were perfused with 1000U/ml Collagenase XI (Sigma-Aldrich) and incubated for 15 minutes at 37°C. Pancreata were manually disrupted and passed through a 70μM filter (Corning) and islets were hand-picked. Isolated islets were allowed to recover for 4–6 hours prior to use in experiments. Islets were cultured in RPMI 1640 supplemented with 10% FBS, 50U/ml Penicillin/Streptomycin and 2mM L-Glutamine at 37° in a 5% CO₂ humidified incubator with or without 1.6ng/ml IFNγ, 0.25ng/ml Il-1β and 0.16ng/ml TNFα (eBioscience) as indicated.