Percpcy5.5 anti cd66b

For flow cytometry, Alexa Fluor 488/anti-CD11b, APC or Alexa Fluor 700/anti-CD33, BV605/anti-CD3, BV510/anti-CD19, PerCPCy5.5/anti-CD66b, PE/Cy7/anti-CD14, PE/Dazzle594/anti-HLA DR (all from Biolegend); APC-eFluor 780/anti-CD11b, PE/anti-TLR2, Alexa Fluor 488/anti-TLR4 (all from Thermo Fisher Scientific) and APC/anti-IL27R from R&D systems were used. For live/dead staining we used LIVE/DEAD™ Fixable Aqua Dead cell stain, and for reactive oxygen species CellROX™ Deep Red Reagent, both from Thermo Fisher Scientific. For immunoblotting, anti- IL-27A (Abcam), phosphorylated (p) NF-κB, NF-κB, STAT1 and pSTAT1 (all from R&D Systems), STAT3 (Novus) pSTAT3 and GAPDH (Cell Signaling Technology) were used.

Antibodies used for flow cytometry were Alexa Fluor488-anti-CD11b, PerCP-eF710/PE/or PE-Dazzel594-anti-HLA DR (clone L243), eF450-anti-HLA DR (clone LN3) PE/Cy7-anti-CD14, APC-anti-CD33, BV510-anti-CD3, BV605-anti-CD19, PerCP-Cy5.5-anti-CD66b, Alexa Fluor700 or PerCP-Cy5.5-anti-IFNγ, APC/Cy7-CD38, PerCP-Cy5.5-anti-CD69, PE-anti-CX3CR1, -anti-B7-H4, -anti-PD-L1, -anti-B7-H3, -anti-PD-L2, -anti-ICOS-L (all from Biolegend, San Diego, CA); FITC- or APCeF780-anti-CD4, Alexa Fluor700- or eF450-anti-CD25, PE-anti-FoxP3 and eF450-anti-IL10 (all from eBioscience); PE-anti- phospho-Zap70^(pY292), PE-anti-phospho-Akt^(pS473) from BD Biosciences. For neutralization studies, mAb to IL-10, IL-6 or rat IgG isotype (10 μg/mL, Biolegend) and polyclonal goat anti-IL-27 or normal goat IgG control (10 μg/mL R&D Systems) control were used.