Anti il 6 pe

For analysis of cell surface markers, the cells were stained with anti-CD86-FITC or anti-CD86-PE, anti-MHC class II-PE-Cy5 (all from BD Biosciences), and F4/80-FITC (Biolegend) or F4/80-APC (eBioscience). Transfection plasmids and siRNA had fluorescent reporters (GFP for plasmid and Cy3-labeled RNA for siRNA), which were used to gate on transfected cells. Macrophages were analyzed for expression of surface markers (MHC class II, CD86, F4/80) or intracellular markers (Egr2, IL-6, TNF). FcRs were blocked with mAb specific for mouse FcR (2.4G2; BD Biosciences). Intracellular staining for Egr2, IL-6 or TNF was performed similarly as described earlier (32 (link), 33 (link)) using fixation/permeabilization agent (eBioscience) and anti-Egr2-APC (eBioscience), anti-IL-6-PE, or anti-TNF-PE mAbs (both from BD Biosciences). The cells were analyzed using LSRFortressa^TM cytometer (BD Biosciences) and FlowJo software (Tree Star Inc.) as we described earlier (34 (link)).

IL-6, IL-1b and TNFα were measured in monocytes (CD14⁺) following the standard intracellular protein staining protocol (BD) using a FACS Calibur (BD) flow cytometer. Briefly, heparinized whole blood was treated with brefeldin A solution (BD) for 4 h, to block cytokine excretion from cells. Erythrocytes were lysed (Cell Lysis Solution 1:10, BD), removed, and white cells were stained with monocyte-specific anti-CD14-APC surface antibody (Biolegend). Where applicable (positive controls), LPS stimulation was performed over 4 h, using bacterial LPS from E. coli (Sigma) at a final concentration of 1 µg/mL. In the next step, cells were permeabilised (Permeabilising Solution 2, 1:10, BD), and treated with intracellular anti-IL-6-PE, anti-IL-1b-FITC and anti-TNFα-PerCP-Cy5.5 antibodies (BD). Antibodies had been titrated before use. Cells were measured immediately following staining and relative fluorescence intensity was quantified against isotype control. For quality control, Hick-3 cytokine positive control cells (BD) were used in every run, as well as signal compensation had been successfully performed prior to each analysis.