Pe conjugated anti cd86

The RAW264.7 cells and peritoneal macrophages (1 × 10⁶/well) were stimulated by heat-inactivated CP (200 μg/ml) or CP-PGN (50 μg/ml) for 24 hours, cell subpopulation markers CD86 of M1 macrophages was analyzed by flow cytometry to evaluate the different phenotypes. The cells were collected and washed with PBS. Cells were stained with PE-conjugated anti-CD86 (0.3 μg/1 × 10⁶ cells, Miltenyi Biotec) for 30 min in the dark. After washing, the expression levels of CD86 of cells were analyzed with a CyAn ADP 9 C flow cytometer (Beckman Coulter). Parallel sets of cells were incubated with monoclonal immunoglobulin isotype control antibody (Miltenyi Biotec) and the fluorescence intensity of these cells served as non-specific negative control. Data were analyzed using FlowJo software (TreeStar, Ashland, OR).

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR; phycoerythrin (PE)-conjugated anti-CD86; and allophycocyanin (APC)-conjugated anti-CD83 (Miltenyi Biotec). APC-conjugated anti-CD3; and Alexa Fluor 488-conjugated anti-IFN-γ (BD Pharmigen). PE-conjugated anti-IL-10; Alexa Fluor 488-conjugated anti-IL-17A; peridinin-chlorophyll-protein (PerCP)-conjugated anti-CD4; Alexa Fluor 488-conjugated anti-FOXP3; PE-conjugated anti-CD127; and APC-conjugated anti-CD25 (Biolegend). Cells were washed with PBS/EDTA 2 mM/0.5% BSA and stained for 15 min at room temperature in the darkness. For analysis of FOXP3 expression in human T cells primed with hmoDCs, cells were first subjected to surface staining with anti-human CD127-PE, CD4-PerCP, and CD25-APC antibodies. After fixation and permeabilization, cells were stained with anti-human FOXP3-Alexa Fluor 488, according to manufacturer’s recommendations. For each staining, the corresponding isotype controls (IgG2A-FITC, IgG1-Alexa Fluor 488, IgG1-PE, IgG2A-PerCP, or IgG1-APC) were also assayed. Flow cytometry analysis was performed in a FACSCalibur in the Cytometry and Fluorescence Microscopy Unit at Complutense University of Madrid.