Anti cd11c pe

The following reagents and fluorochrome-conjugated monoclonal antibodies were used in this study for T cell phenotypic and functional analyses: PKH26 membrane dye (Sigma), APC- anti-CD4, APC-anti-CD8a, PE-anti-CD4, PE-anti-CD25, FITC or APC-anti-TNFα, PE-anti-ICOS-L FITC-Annexin-V (all from eBioscience), FITC-anti-CXCR4, APC-anti-CXCR7, PE-anti-hCCR4 (all from R&D Systems), and FITC-anti-CD279/PD-1 (Biolegend, San Diego, CA). Intracellular expression of TNFα was detected following fixation and permeabilization (fixation/permeabilization buffer concentrate, Cat. No. 00-5123-43, diluent, Cat. No. 00-5223-56, and permeabilization buffer, Cat. No. 00-8333-56, all from eBioscience). All samples were analyzed with a FACSCalibur flow cytometer, using CellQuest software (BD Biosciences). For analysis of tumor ascites CD14⁺ cell phenotype, the following monoclonal antibodies were used: FITC-anti-CD14, PE-anti-CD11b (both from R & D Systems), PE-anti-CD11c, PE-anti-B7-H1, PerCP-anti-CD14 (all from BD Biosciences), FITC-anti-CD279/PD-1, FITC-anti-CD83, FITC-anti-CD80, PE-anti-CD86, APC-anti-CD14, and PE-IL-1β (all from eBioscience). Intracellular expression of IL-1β was assayed following overnight incubation of CD14⁺ cells in the presence of Brefeldin A, followed by fixation and permeabilization as described above.

Peritoneal exudate cell (PEC) suspensions were blocked with Fc Block (BD Biosciences, San Jose, CA, USA) and subsequently stained with specific Abs including: anti-Ly6G FITC, anti-CD11cPE, anti-CD11b PerCP/CY5.5, Siglec-F PE, anti-c-kit APC, F4/80 APC, anti-CD4APC, anti-CD19 PerCP/Cy5.5, CD3PE (BD Biosciences, San Jose, CA, USA) and anti-CD206PE or anti-CD206 Alexa fluor 488 (Biolegend, USA). Cells were collected 4 hours after microparticle inoculation for analysis of phosphorylation of BTK (Y-551) or SYK (Y-348). Intracellular staining was performed using anti-mouse phospho-BTK/ITK (Y551/Y511) PE (eBiosciences, San Diego, CA, USA), anti-mouse phospho-SYK (Y-348) PE antibody (BD Biosciences, San Jose, CA, USA) and FOXP3/Transcription Factor Staining Buffer Set, which was used as per the manufacturer instructions (eBiosciences, San Diego,CA, USA). For CFSE-labeled cells, anti-CD4 PerCP (BD Biosciences, San Jose, CA, USA), and KJ1–26 PE (BD Biosciences, San Jose, CA, USA) were used to phenotype DO11.10 T cells and cell cycle progression was examined as previously discussed^{10 (link)}. For FACS analysis of whole synovial tissues, samples were digested at 37°C for 1 hour with 0.1% collagenase in RPMI supplemented with 10% FBS, 1000U/ml penicillin, 1000U/ml streptomycin, and 2nm L-glutamine. Single cell suspensions were then prepared and blocked and stained as described above.