Anti mouse cd8 fitc

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.

To determine the expression of CD8 + CD28-T cells in mice, FCM analysis was performed on isolated spleen cells using anti-mouse CD3, CD8, and CD28 monoclonal antibodies. The spleens were minced mechanically and lysed in a lymphocyte separation medium. The isolated spleen cells were washed and resuspended in PBS. Anti-mouse CD3 APC, anti-mouse CD8 FITC, and anti-mouse CD28 PE (eBioscience, San Diego, USA) were mixed at 4 0 C for 10 min in the dark. And then, the cell suspension was analyzed using FCM.