B10 br mice

CD4⁺ T cells were purified from the spleens of B10.BR mice (Jackson Laboratory) using the EasySep™ Mouse CD4⁺ T Cell Isolation Kit (STEMCELL Technologies) following the manufacturer’s instructions. Cells were then labelled with 5 μM CFSE (carboxyfluorescein succinimidyl ester) according to the manufacturer’s instructions (Invitrogen) before being co-cultured with bone-marrow-derived dendritic cells at different T-cell/dendritic cell ratios (1:5, 1:10, 1:20) for 72 h in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum, 50 μM β-mercaptoethanol and 0.2% primocin. Cells were then harvested and lymphocytes were labelled for flow cytometry analysis.

Female NOD/ShiLt, NOR/LtJ, C57BL/6, and B10.BR mice were purchased at three weeks of age from Jackson Laboratories (Bar Harbor, Maine). All animal studies were performed in accordance with the guidelines of the Yale University Institutional Animal Care and Use Committee. All mice were housed in microisolator cages with free access to food and water and maintained under the following facility conditions: temperature: 72 °C ± 2 °C; humidity: 50% ± 30%; 12:12 light/dark cycle (i.e., 12 h of light from 7 a.m.–7 p.m. then 12 h of dark from 7 p.m.–7 a.m.). Glucose levels were measured in blood withdrawn from the retroorbital sinus of anesthetized mice using Lite glucometer and test strips (Abbott Laboratories, Abbott Park, IL, USA). Mice with blood glucose values greater than 250 mg/dL were considered diabetic. Serial serum samples were acquired from NOD and age matched control animals. Pancreas and liver tissue extract were prepared in RIPA buffer containing a mixture of protease inhibitors (Complete protease inhibitor, Roche Diagnostics).