Islets were isolated from C57BL/6 mouse pancreas using the collagenase digestion method. Briefly, Ivacaftor chemical structure the organs were minced into smaller pieces and subsequently incubated with collagenase
type V solution (1 mg/ml; Sigma, St Louis, MO, USA) in Hanks’s balanced salt solution (HBSS) at 37°C for 10–15 min with vigorous shaking. Following cold HBSS addition to stop the digestion, the islets were handpicked and seeded into 96-well flat-bottomed plates (30/well) in culture medium (RPMI-1640 + 0·5% FCS). After overnight rest, pancreatic islets were treated with apoTf (25 µg/ml) and added to the proinflammatory cytokine cocktail for the next 24 h to be then analysed for cell viability. The viability of RINm5F cells, as well as of pancreatic
islets, was assessed using the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan. Cells were washed with phosphate-buffered saline (PBS) to remove non-adherent dead cells, and MTT (0·5 mg/ml) was added to the remaining adherent cells. Pancreatic islets were then collected, centrifuged learn more and the pellets dissolved in MTT solution for 60 min at 37°C. After incubation, dimethyl sulphoxide (DMSO) was added to the adherent insulinoma cells, or pellets of pancreatic islets to dissolve the formazan crystals. Absorbance was finally measured at 570 nm wavelength, with a correction at 690 nm, using an automated microplate reader (LKB 5060-006; LKB, Vienna, Austria). The results of the MTT assay are presented as the proportion of control values obtained in untreated cell cultures. Data are expressed as the mean ± standard deviation (s.d.) of values obtained in at least five, three and seven individual experiments for mouse pancreatic islets or RINm5F cells, respectively. All animal experiments were
conducted in accordance with national and local regulations regarding animal welfare, and with the approval of the institutional animal care and use committee (IACUC). Female non-obese diabetic (NOD) C59 mice (8–9 weeks old) and male diabetes-prone (DP) BB rats (5–6 weeks old) were purchased from Charles River, Milano (Italy). Rodents were housed under standard conditions with ad libitum food and water at the University of Catania (Italy). All animals were housed for 1 week prior to study initiation and then randomized into five per cage corresponding to one specific experimental condition. NOD mice and DP-BB rats were screened for glycosuria twice per week starting at the ages of 8–9 and 5–6 weeks, respectively. Two animal models of type 1 diabetes were used for these experiments. First, male DP-BB rats (36–45 days of age) were divided into four groups (n = 14 per group) to be treated with human apoTf at different concentrations (1·25, 2·5 or 5 mg/kg) or PBS for 7 consecutive weeks.