(f) n = 6 per group. To further investigate the increased GSIS assays. animal care and use committees. Blood glucose measurements were taken weekly from 4 weeks of age up to 35 weeks of age using a blood glucose meter (AlphaTRAK) and rat/mouseCspecific test strips. At the end of the study, mice were subjected to collagenase perfusion of the pancreas to isolate pancreatic islets for analysis or pancreatic dissection for either cryofixation or paraffin embedding. A separate cohort of mice from the 17-week study was euthanized at the 4- to 5-week time point, and pancreata were collected for sectioning. MLD-STZ induction of diabetes MLD-STZ (Sigma; #S01230) [50 mg/kg of body weight (BW)] induction of hyperglycemia and treatment with 10 g/kg BW Ex4 TH-302 (Evofosfamide) (Sigma-Aldrich; #E7144) was conducted as previously described (16). Mouse islet isolation and GSIS assay Mice were euthanized using 2,2,2-tribromoethanol (Sigma; #{“type”:”entrez-nucleotide”,”attrs”:{“text”:”T48402″,”term_id”:”650382″,”term_text”:”T48402″}}T48402) anesthesia followed by cervical dislocation. Intact pancreatic islets were isolated from mice using a collagenase digestion protocol (23). On CLC the day of isolation, islets were picked into 100 L of islet medium (RPMI 1640; TH-302 (Evofosfamide) Gibco; #11879020) containing 11.1 mmol/L glucose (Fisher Scientific; #D16), 10% heat-inactivated fetal bovine serum (Sigma-Aldrich; #12306C), and 1% Hepes (Sigma; #H4034) and penicillin/streptomycin (Gibco; #15070-063)) in each well of a 96-well V-bottom tissue culture-coated plate (Corning Life Sciences; #3894) according to a protocol optimized in our laboratory (24). Insulin enzyme-linked immunosorbent assays were performed essentially as described elsewhere (11). Glucose tolerance testing Animals were fasted for 4 to 6 hours before glucose tolerance testing. Glucose was given using an oral gavage at a dose of 2 g/kg BW. Blood glucose measurements were taken immediately before gavage and at 5, 15, 30, 60, and 120 minutes following TH-302 (Evofosfamide) gavage. For female mice, blood was collected using a lateral tail nick at baseline and 5 minutes after gavage. Oral glucose tolerance testing was performed at 16 and 24 weeks of age for male and female mice, respectively. Whole pancreas staining For all slide staining assays, 10-m serial sections were cut on positively charged slides, with 18 sections per stop position (three per slide) and three stop positions per pancreas separated by at least 200 m. Immune infiltration of the (gene symbol: (((clone 145-2C11) and 1 g/mL soluble anti-CD28 (clone 37.51) (BD Biosciences; #553057 and #553294) in complete RPMI media with 10% fetal bovine serum. Suspensions of single cells were incubated with GolgiStop (BD Biosciences; 51-2092KZ) for 4 hours before staining. Cells were stained for surface markers, fixed and permeabilized with Cytofix/Cytoperm Plus reagents (BD Biosciences; #51-2090KZ and #51-2091KZ), stained for intracellular cytokines, and analyzed with the BD LSR II flow cytometer. Fluorescent antibodies for CD3(clone 145-2C11), CD8(clone 53-6.7), TNF(clone MP6-XT22), and IFN(clone XMG1.2) were purchased from BD Biosciences (#563565, #561092, #561041, and #563376, respectively). The fluorescent antibody for CD4 (clone RM4-5) was purchased from eBioscience (#11004282). Ghost Dye viability reagent was purchased from Tonbo Biosciences (#130865). Flow cytometry data were analyzed with FlowJo software. Statistical analysis Data are expressed as mean standard error of the mean unless otherwise noted. Data were compared by one- or TH-302 (Evofosfamide) two-way analysis of variance or Student test as appropriate and as described in the figure legends. A value 0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism version 6 (GraphPad.

(f) n = 6 per group