Cmrl 1066 medium

Human islets were provided through the Uppsala facility for the isolation of human islets from Scandinavian brain-dead individuals. Human islets were cultured free-floating in Sterilin dishes in CMRL 1066 medium (ICN Biomedicals, Costa Mesa, CA, USA) containing 5.6 mmol/l glucose, 10% FCS and 2 mmol/l l-glutamine for 1–5 days prior addition of adenosine and inosine. Cell viability was assessed by PI and Hoechst staining followed by fluorescence microscopy.

Human EndoC-βH1 cells were cultured as previously described (22 (link)). Mouse insulinoma (MIN6) cells were cultured in 25 mmol/L glucose DMEM supplemented with 15% FBS. Palmitate (sodium salt, Sigma-Aldrich) exposure media were supplemented with 2% fatty acid free BSA (Roche). During incubations with Palmitate serum-free medium was used for MIN6 cells. KRBH buffer contained 115 mmol/L NaCl, 24 mmol/L NaHCO_3, 5 mmol/L KCl, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 0.2% BSA, and 10 mmol/L HEPES. Human pancreatic islets were kindly provided by Professor Olle Korsgren (Department of Radiology, Oncology and Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden), through the Uppsala facility for the isolation of human islets from Scandinavian brain-dead individuals. After isolation, the islets were cultured free-floating in Sterilin dishes in CMRL 1066 medium (ICN Biomedicals, Costa Mesa, CA, USA) containing 5.6 mmol/L glucose, 10% fetal calf serum, and 2 mmol/L L-glutamine for 1–5 days, and then subsequently transferred to the same culture conditions as those used for Palmitate exposure of EndoC-βH1 cells. All cells were kept at 37 °C in a humidified atmosphere with 5% CO₂.
Etomoxir was from Sigma-Aldrich. GPR40 antagonist (GW1100) was from Calbiochem. The PKC inhibitor Bisindolylmaleimide (GF109203X) and the PKD inhibitor CID755673 were from Tocris Bioscience (Bristol, UK).

Human pancreatic islets were kindly provided by Nordic Network for Clinical Islet Transplantation. Only organ donors who had agreed to donate for scientific purposes were included. Informed written consent to donate organs for medical and research purposes was obtained from donors, or relatives of donors, by the National Board of Health and Welfare (Socialstyrelsen), Sweden. Permission to obtain pancreatic islet tissue from the Nordic Network for Clinical Islet Transplantation was reviewed and approved by the local ethics committee (Regionala etikprövningsnämnden, Uppsala) in Uppsala, Sweden.
After isolation, the islets were cultured free-floating in Sterilin dishes in CMRL 1066 medium (ICN Biomedicals, Costa Mesa, CA, USA) containing 5.6 mM glucose, 10% fetal calf serum and 2 mM L-glutamine for 1–5 days. All cells were kept at 37°C in a humidified atmosphere with 5% CO₂. Human EndoC-βH1 cells were cultured in the presence of 2% fatty acid free bovine serum albumin as previously described [24 (link)].