Pe conjugated anti f4 80 antibody

Whole blood obtained cardiac puncture was buffered with EDTA, subjected to red-cell lysis using BD FACS Lysing Solution (BD Biosciences), and washed in PBS supplemented with 3% fetal calf serum. Blood leukocytes were first incubated with rat anti-mouse FcR/III antibody (2.4G2) (BD Biosciences) to minimize non-specific binding of antibody to FcR. They were further incubated with APC-conjugated anti-CD11b antibody (BD Biosciences) and PE-conjugated anti-F4/80 antibody (BD Biosciences) for 10 min on ice, and washed with PBS supplemented with 3% fetal calf serum. The percentages of CD11b + and F4/80 + cells were analyzed by the FACS Canto II flow cytometer (BD Biosciences) using EXPO32 software (Beckman Coulter).

For quantification of GFP+ cells in circulation, mouse blood was withdrawn from the tail and subjected to flow cytometry analysis based on direct fluorescent GFP in Becton flow cytometry machine (BD Biosciences, Shanghai, China). The number of the positive cells was automatically obtained by machine counting. For isolation of macrophages in the tumor, dissected tumor was digested with 0.25% Trypsin (Roche) for 30 minutes before all dissociated cells were incubated with PE-conjugated anti-F4/80 antibody (BD Biosciences) for 15 minutes to label all macrophages for sorting.