Mcp 1 apc

Zymosan A from Saccharomyces cerevisiae was purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-mouse mAbs, such as CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650, MHC II (I-A/I-E)-PECy5, Siglec-F-PE, LY6G-APC, LY6C-APC-CY7, IL-6-PE and MCP-1-APC were purchased from eBioscience (San Diego, CA, USA). Recombinant mouse B7-H1/Fc chimera (1019-B7) was purchased from R&D Systems (Minneapolis, MN, USA). Human IgG for control experiments was purchased from Sigma–Aldrich. Anti-mouse PD-1 (clone J43) were purchased from eBioscience. LY294002, PD98059, SP600125 and SB203580 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). The following mAbs were used for western blotting and immunoprecipitation: anti mPD-1 (RPMI-30; eBioscience), anti-phosphotyrosine Ab (Cell Signaling Technology, Beverly, MA, USA), anti-SHP-1 (sc-287), anti-SHP-2 (sc-280), p-STAT1, STAT1, p-STAT6 and STAT6 (Cell Signaling Technology), anti-mouse IgG-HRP, anti-rabbit IgG-HRP Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Cell suspensions were stained with violet live/Dead cell stain kit (Life Technologies, Eugene, OR, USA) for the analysis of viability, and then washed and stained with appropriate anti-mouse mAbs CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650, MHC II (I-A/I-E)-PECy5, Siglec-F-PE, LY6G-APC and LY6C-APC-CY7 (all from eBioscience) for 30 min. For the expression of PD-1, bone marrow–derived macrophages (BMDMs) were collected from cultures and washed with FACS buffer. And then, the cells were stained with PE-conjugated anti-mouse PD-1 (clone J43). For the intracellular staining of macrophages from peritoneal cavity, cells were stained with violet (live/Dead) and membrane markers (CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650), and then permed with perming buffer (BD Biosciences, San Jose, CA, USA), continued with staining of intracellular cytokines (IL-6-PE and MCP-1-APC, both from eBioscience). Data were acquired on BD fluorescence-activated cell sorting LSR-II (BD Biosciences) and analyzed with Flowjo software (TreeStar, Ashland, OR, USA).