Figure 7. APC directly binds Ly6C⁺ cells.

(A) The expression of PAR-1 on the surface of Ly6C⁺ cells is assessed by flow cytometry. Left panel shows the histogram representation of PAR-1 expression on Ly6C⁺ cells. Right panel shows the graphical representation of MFI corresponding to PAR-1 expression on Ly6C⁺ cells. (B) To determine whether APC can directly interact with Ly6C⁺ cells, fluorochrome-conjugated APC were incubated with splenic cells, and the binding of APC to Ly6C⁺ cells was assessed by flow cytometry (gated on Ly6C⁺ cells). (C) To examine whether anti-PC can decrease the interaction of APC to Ly6C⁺ cells, fluorochrome-conjugated APC were incubated with splenic cells with or without anti-PC, and the binding of APC to Ly6C⁺ cells was assessed by flow cytometry. (D) To examine whether APC utilizes the CD11b integrin to bind to Ly6C⁺ cells, fluorochrome-conjugated APC were incubated with splenic cells with or without anti-CD11b, and the binding of APC to Ly6C⁺ cells was assessed by flow cytometry. (E) Stat3 expression in splenic CD11b⁺Ly6C⁺ cells was assessed by flow cytometry. Left panel shows the histogram representation of Stat3 expression in CD11b⁺Ly6C⁺ cells from anti-PC and control mice. Right panel is the graphical representation of the MFI corresponding to Stat3 expression in CD11b⁺Ly6C⁺ cells. Data are represented as means ± SEM (n = 4; * p < 0.05 by Student t-test). (F) The phosphorylation status of Stat3 in Ly6C⁺ cells was assessed by flow cytometry. Levels of phosphorylated Stat3 (pStat3) in Ly6C⁺ cells from anti-PC and control mice are represented as flow cytometric histograms (left panel) and as mean MFI ± SEM (right panel, n=3; p calculated by Student t-test). (G) Apoptotic CD11b⁺Ly6C⁺ cells from both anti-PC or IgG mice was assessed by annexin V binding. The degree of annexin V binding to CD11b⁺Ly6C⁺ cells was examined by flow cytometry, and levels of annexin V binding are shown as flow cytometric histograms (left panel) and as mean MFI ± SEM (right panel, n =4).