1C, D). Laboratory UR 1102 Animals in Sweden. The protocol was authorized by the Committee within the Ethics of Animal Experiments in the University or college of Gothenburg (Permit Quantity: 338C2012). All attempts were made to minimize animal suffering. Insulitis and beta cell area Sections 200C300 m apart from the pancreas of 4-, 9- and 23-week-old mice UR 1102 were stained with haematoxylin and eosin and obtained according to the UR 1102 following criteria: no infiltration (0); peri-insulitis (1); 50% damage of islet (2); 50% damage of islet (3). We obtained 100 (at 4 and 9 weeks) and 50 (at 23 weeks) islets per mouse. For beta cell area, sections 300 m apart through the whole pancreas were stained with guinea pig anti-insulin Rabbit Polyclonal to ADCK4 (Dako), visualized using VECTASTAIN ABC and Vulcan Fast Red Chromogen Kit 2 (Histolab), and counterstained with haematoxylin. The area stained for insulin was compared with the total area of the cells using Biopix software. Insulin autoantibodies Serum insulin autoantibodies (IAA) were analysed using a competitive radiobinding assay as previously explained [15]. The results were indicated in relative devices (RU) based on standard curves run on each plate. The cut-off value for mouse IAA positivity was arranged in the mean+3SDS in 29 BALB-mice, i.e. 0.88 RU. Cytokine measurements Serum from mice at 15 and 23 weeks was analysed with Mesoscale 9-plex cytokine assay according to the manufacturer’s instructions. Oral glucose tolerance test (OGTT) and insulin measurements Mice aged 17C18 weeks were fasted for 4 h and orally gavaged with 20% D-glucose (2 g/kg). Blood was drawn from your tail vein at 0, 15, 30, 60, 90 and 120 min and blood glucose levels were measured using a HemoCue glucometer. Insulin levels were measured at 0, 15 and 30 min after gavage. In mice that did not progress to diabetes by 30 weeks of age, insulin levels were measured after a 4 h fast. Insulin was measured using insulin ELISA-kit (Crystal Chem). Analysis of polar metabolites by GCGC-TOFMS An established metabolomics platform using two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCGC-TOFMS) was used to analyse polar metabolites in serum [24]. Serum samples (30 l) were combined with 10 l of an internal standard, labelled palmitic acid (16:0C16,16,16d3; 500 mg/l), and 400 l of methanol, vortexed for 2 min and incubated for 30 min at space temp. The supernatant was separated by centrifugation at 5590 for 5 min at space temperature. The sample was dried under a constant circulation of nitrogen. Twenty-five l of 2% methoxyamine hydrochloride in pyridine was added to the dried sample and incubated at 45C for 1 h and then derivatized with 25 l of N-methyl-N-(trimethylsilyl)-trifluoroacetamide by incubating at 45C for 1 h. Five l of retention index standard combination with UR 1102 five alkanes (400 mg/l) was added to the metabolite combination. Sample order for analysis was founded by randomization. The samples were analysed on a Leco Pegasus 4D GCGC-TOF mass spectrometer with Agilent systems 6890N GC and Combi PAL autosampler. Data were processed using the Guineu software [24]. Analysis of molecular lipids by UPLC-MS An established platform based on Acquity Ultra Overall performance LC coupled to time-of-flight mass spectrometry (UPLC-MS) was used to analyse the molecular lipids in aliquots (10 l) of serum samples [25]. The data were processed using MZmine 2 software [26] and the lipid recognition was based on an internal spectral library or on recognition using tandem MS [25]. Statistical analysis Data were analysed by Student’s t test and offered as mean SEM or SD. Survival curves were analysed with log-rank (Mantel-Cox) test. IAA positivity was analysed by Fisher’s exact test. Univariate statistical analysis of metabolomics data used MATLAB r2012a. Clustering was performed.