Low endotoxin and fatty acid free bsa w v

INS-1E cells (generated and kindly provided by P. Maechler and C. Wollheim, University of Geneva, Switzerland [52 (link),53 (link)]) were cultured in complete RPMI-1640 medium with GlutaMAX (Life Technologies, Naerum, Denmark) supplemented with 10% fetal bovine serum (FBS) (v/v), 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 mmol/L Hepes, 50 μmol/L β-mercaptoethanol (all from Life Technologies, Naerum, Denmark) and 1 mmol/L sodium pyruvate (Sigma-Aldrich, Soeborg, Denmark) at 37 °C in a humidified atmosphere with 5% CO₂. For GLT treatment, culture medium with 1% FBS (v/v) and 1% low endotoxin and fatty acid free BSA (w/v) (Sigma-Aldrich, Soeborg, Denmark) supplemented with 25 mmol/L glucose and 0.5 mmol/L ethanol-dissolved palmitate (both from Sigma-Aldrich, Soeborg, Denmark) conjugated to BSA at a molar ratio of 3.33:1 was used. Ethanol and BSA was used as vehicle for GLT. All experiments were performed within the passage numbers 55–80, since INS-1E display a stable phenotype between passages 40–100 [53 (link)]. All cells tested negative for Mycoplasma.

Pancreata were inflated and digested with 1.4 mg/mL collagenase type 4 (Worthington Biochemical Corporation, Lakewood, CA, USA), and incubated for 30 min at 37 °C in a water bath. Islets were filtered, handpicked to purity and cultured at 37 °C in RPMI-1640 with 11.1 mM glucose supplemented with 10% FBS (v/v), 2 mmol/L GlutaMAX, 100 IU/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL gentamycin (all from ThermoScientific, Zug, Switzerland). Islets were exposed to 0.5 mmol/L palmitate and 25 mmol/L glucose in medium with 1% low endotoxin and fatty acid free BSA (w/v) (all from Sigma-Aldrich, Buchs, Switzerland) and 1% FBS (v/v) for 48 h. All animal experiments were performed according to the Swiss veterinary law and institutional guidelines, and upon approval (accessed on 2 January 2017) by Swiss authorities (Veterinäramt, Basel-Stadt, Switzerland) within the framework of the animal experiment permit 2401.