Characterization and phenotype of mouse monocytes. (A) Monocytes were identified in lysis buffer-treated, B-cell-depleted, heparinized blood by correlating their expression of CD11b, Gr-1, F4/80, and Ly-6C. Monocytes and neutrophils are shown in the boxes with solid and dashed lines, respectively, to allow the comparison of their relative expression level of the indicated markers. Cells in the boxes with double dashed line correspond to noncirculating CD11b^high F4/80^high macrophages from the thoracic cavity. (B) FSC versus SSC profiles of ungated total blood cells (left), and after gating for monocytes on the basis of their CD11b versus Ly-6C expression (right). (C) CD11b versus Ly-6C profiles of total bone marrow samples and of purified bone marrow monocytes (see “Materials and methods” for details on monocyte purification). (D) Phenotypic analysis by flow cytometry of C57BL/6 mouse blood monocytes performed by triple immunofluorescent staining after gating on monocytes on the basis of their CD11b versus Ly-6C profile, as shown in panel A. The percentage of cells with a fluorescence intensity over the vertical lines (dark gray area of the profiles), corresponding to the upper limit of control background staining (shown in the CD11b histogram), is indicated. White profiles on the CD43 and CD24 histograms correspond to the expression of these markers by BALB/c blood monocytes (in these cases the percentage of cells with a fluorescence intensity over the background staining is indicated in parentheses). Data are representative of 5 experiments with similar results. (E) RT-PCR analysis of TLR-4 and TLR-9 expression by bone marrow monocytes. CD8⁺ and CD8^- splenic DCs were used as positive controls for both TLR-4 and TLR-9 expression. β-Actin mRNA levels are shown to control for the relative expression of TLR-4 and TLR-9 mRNA in the different populations considered. Data are representative of 2 experiments with similar results.