ELISAs were developed using o-phenyl diamine dihydrochloride (OPD) substrate (Sigma) in sodium citrate buffer, Rapamycin cost pH 5, plus H2O2. H2SO4 (12·5%) was used to stop the OPD reaction, and plates were read at
490 nm using Softmax™ Pro software (MDS Analytical Technologies, Sunnyvale, CA). Modulation of the CD3–TCR complex in peripheral blood was analyzed by flow cytometry 2 and 24 hr after each dose when mice were dosed every 24 hr and 2 and 72 hr after each dose when mice were dosed every 72 hr. Following red blood cell lysis, cells were stained using murine antibodies to CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7) and TCR-β (H57-597) (BD Biosciences, San Jose, CA). Molecules of equivalent soluble fluorochrome (MESF) values were generated MK-2206 cell line using Quantam™ fluorescein isothiocyanate (FITC) MESF microspheres as per the manufacturer’s instructions (Bangs Laboratories,
Fisher, IN). FoxP3 expression was evaluated using a FoxP3 staining kit (NRRF30 clone; eBioscience, San Diego, CA), as per the manufacturer’s instructions. Fluorescent cells were analyzed by flow cytometry using FACScalibur (BD Biosciences). In Study B, serum was collected before and after treatment and analyzed for the murine C-peptide I content by ELISA, according to the manufacturer’s instructions (ALPCO, Salem, NH). In Study B, pancreata were fixed in formalin, processed and embedded in paraffin. CYTH4 Sections of 4–5 μm in thickness were stained with haematoxylin and eosin. Islet inflammation was evaluated using light microscopy by a board-certified veterinary pathologist (Charles River Laboratories, Wilmington, MA). Peri-insulitis inflammation was scored as: 0 = normal (no leucocytes);
1 = minimal (< 5 leucocytes in any islet); 2 = mild (6–20 leucocytes in the ‘most severe’ islet); 3 = moderate (21–50 leucocytes in the ‘most severe’ islet); 4 = marked (> 50 leucocytes in the ‘most severe’ islet); or 5 = severe (> 50 leucocytes in > 1 islet). MESF values were analyzed using repeated-measures analysis of variance (anova), with treatment and time as factors. Lymphocyte count data were analyzed by one-way anova. Pairwise treatment group comparisons for these analyses were carried out using the corresponding t-tests. Fisher’s exact test was used for pairwise treatment group comparisons of proportion data. Exploratory comparisons between post-treatment remission and diabetic groups were made using t-tests (quantitative data), Fisher’s exact test (proportion data), or the chi-square test (categorical data). P-values were not adjusted for multiple comparisons.