Anti mouse cd4 percp

A total of 1 × 10⁶ splenic T lymphocytes in vivo and three kinds of in vitro cocultured T cells described above were separately washed and resuspended in FACS buffer containing phosphate-buffered saline (PBS) and 0.5% bovine serum albumin (BSA). The suspension was incubated with fluorescently labeled monoclonal antibodies for 1 h at 4 °C in the dark. Anti-mouse PerCP-CD4 (#46-0041-82, Thermo Fisher Scientific, Waltham, USA) and anti-mouse PE-IL-17 (#12-7177-81, Thermo Fisher Scientific, Waltham, USA) monoclonal antibodies were prepared to identify Th17 cells, while Anti-mouse PerCP-CD4, anti-mouse APC-CD25 (#17-0251-82, Thermo Fisher Scientific, Waltham, USA), and anti-mouse PE-Foxp3 (#12-5773-82, Thermo Fisher Scientific, Waltham, USA) monoclonal antibodies were prepared to identify Tregs.
After washing three times, the cells were resuspended again in PBS containing 0.1% BSA for flow cytometry (#Canto II, BD Biosciences, New Jersey, US). The data were analyzed with FlowJo (#V10.5, BD Biosciences, New Jersey, US).

For detecting macrophage/monocytes, DCs, neutrophils^{61 (link)} and CD8 T cells^{62 (link)}, 0.5 × 10⁶ cells were stained with anti-mouse CD8-FITC, anti-mouse CD49b-FITC, anti-mouse CD11b-phycoerythrim (PE), anti-mouse CD11c-APC-Cy7 (for DC exclusion) or CD11c-Per-CP (for activated DC population analysis) and anti-mouse Ly6G-APC-Cy7. To detect activated macrophage and DC populations, anti-mouse CD40-APC or OX40L-APC antibodies were also added (all from eBioscence). To detect activated T cells, anti-mouse CD3-PE, anti-mouse CD4-PerCP, anti-mouse CD8-FITC and anti-mouse OX40-APC or anti-mouse CD25-APC antibody (all from eBioscience) staining was performed for 30 min at 4 °C after blocking with 10% FCS in PBS with 10 mM EDTA, then fixed with 2% paraformaldehyde in PBS with 10 mM EDTA. Cells were analysed using FACS Canto (Becton Dickinson) acquiring 20,000 leukocytes (gated according to forward and side scatters with doublets excluded according to FCS-A/FCS-H dot plots). FACS results were subsequently analysed with FACS Diva (Becton Dickinson). Corresponding unstained cell samples for each antibody were acquired to establish gating areas for positively staining cells.

Cell pellets were prepared from spleen tissues. The regulatory T cell populations were examined using anti-mouse CD4–peridin chlorophyll protein (perCP) and anti-mouse CD25-allophycocyanin (APC) (eBioscience); then, the cells were fixed and permeabilized using a Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For Th17 cell analysis, before FACs staining, the cells were stimulated with 25 ng/mL phosphomolybdic acid (Sigma-Aldrich, St. Louis, MO, USA), 250 ng/mL ionomycin (Sigma-Aldrich), and Golgi Stop (BD Biosciences, San Diego, CA, USA) in 5% CO₂ at 37°C for 4 hours. The cells were stained with anti-mouse CD4 PerCP, and then with an anti-mouse IL-17 FITC (eBioscience), followed by fixation and permeabilization using a Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions. The samples were analyzed using a FACSCalibur instrument (BD Pharmingen; BD Biosciences).