Protocols for Culturing and Analyzing Pancreatic Islets

Human islets with >90% purity and viability were obtained from non-diabetic de-identified cadaveric donors through the Integrated Islet Distribution Program (IIDP). The characteristics of the donors are reported in Supplementary Table 1. Upon receipt, the islets were cultured as described^{70 (link)}. Murine islets of Langerhans were isolated as previously described^{31 (link)}. Procedures on rodents have been performed according to guidelines and regulations approved by the Einstein Animal Care and Use Committee. INS-1 cells were maintained in monolayer culture in RPMI-1640 medium, as previously described by our group^{71 (link)}. Insulin levels were determined as described and validated^{31 (link),71 (link)–73 (link)}. In some experiments the cells were treated with glucose (5.5 and 16.7 mM, Bio-Techne, Abingdon, UK), sirolimus (LC Laboratories, Woburn, MA, dissolved in dymethylsulfoxide), or L-leucine (10 mM, MyBioSource, San Diego, CA, USA) and glutamine (2 mM, MyBioSource). Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT colorimetric assay, spectophotometrically (570 nm) measuring the ability of metabolically active cells to reduce MTT.