Anti mouse foxp3 pe or isotype control mabs

As previously described [12 (link)], CD4⁺CD25⁻T cells were purified from the spleens of D1 mice using a CD4⁺CD25⁺ regulatory T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and were subsequently stimulated with anti-CD3/CD28 monoclonal antibodies (mAbs, 50 ng/mL, PeproTech) in the presence of TGF-β1 (5 ng/mL, PeproTech) for 5 days. The T cells were incubated with anti-mouse CD4-FITC mAb, fixed, permeabilized, and stained with anti-mouse FoxP3-PE or isotype control mAbs (BD Biosciences Pharmingen). And the suppressive activity of these cells against T cell proliferation was examined using an in vitro suppressive assay. Approximately 3 × 10⁶ cells were transferred to CIA mice at 25 days after the first CII immunization.

iTregs were derived from CD4⁺ CD25⁻ T cells that were purified from the splenocytes of D1 mice using a CD4⁺ CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec, Germany) and stimulated with an anti-CD3/CD28 mAb (50 ng/ml, PeproTech) in the presence of TGF-β1 (5 ng/ml, PeproTech) and IL-2 (100 ng/ml, PeproTech) for 5 days [8 (link)]. Then, iTreg_mtDC or iTreg_mDC were generated by expanding iTregs for 4 days using mtDCs or mDCs at a 5 : 1 ratio (5 T cells : 1 DC). The different types of iTregs were collected, counted, incubated with anti-mouse CD4-FITC and CD25-PE-Cy5 mAbs, fixed, permeabilized, and stained with antimouse Foxp3-PE or isotype control mAbs (BD Biosciences Pharmingen). Then, the coexpression of CD25 and Foxp3 was determined by flow cytometry.