Macrophage Surface Marker and Intracellular Cytokine Analysis

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)).