Anti mouse cd25 monoclonal antibody

Normoglycemic 12- to 14-week-old female NOD mice were used as donors to prepare splenocytes suspension. Female NOD-SCID recipient mice were injected with 2 × 10⁷ splenocytes via the retro-orbital plexus and checked twice a week for development of diabetes. For adoptive transfer to Rag1^−/− mice, effector T cells were isolated from 6- to 8-week-old normoglycemic female NOD mice using Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-mouse CD25 Monoclonal Antibody (eBioscience) according to the manufacturer’s instructions. For isolation of splenic DCs, CD11c-PE (N418; BioLegend) and Anti-R-PE Magnetic Particles–DM (BD Biosciences) were used according to the manufacturer’s instructions. T cells (7.5 × 10⁶) and DCs (7.5 × 10⁵) were mixed together and injected into Rag1^−/− mice via retro-orbital plexus. Mice were checked twice every week for diabetes or sacrificed 1 month after transfer for T cell analysis.

CD4⁺ effector T cells were isolated from transgenic and non-transgenic (non-Tg) mice using Mouse CD4 T Lymphocyte Enrichment Set–DM (BD Biosciences) and anti-mouse CD25 Monoclonal Antibody (eBioscience). T cells were cultured with equal numbers of splenic DCs from wild-type mice, in a 96-well plate. Co-culture was supplemented with proinsulin peptide (Table 2). After 48 h, 1μCi [methyl-³H] thymidine (PerkinElmer, Shelton, CT, USA) was added to each well and 16–18 h later [methyl-³H] thymidine incorporation was measured using a Packard TopCount Microplate Scintillation Counter.