Src/Syk activation by zymosan. IFN-γ-primed bone marrow-derived macrophages were fed zymosan for 15 minutes in the presence of sodium orthovanadate and activation of Syk (A) and Src family (B) kinases was visualized by immunofluorescence microscopy using anti-phospho-Syk (Tyr519/520) and anti-phospho-Src (Tyr 416) antibodies, respectively. (C) Tyrosine phosphorylation of Syk (left panels) and Src family kinases (right panel) was measured by flow cytometry of bone marrow-derived (BMMO) or peritoneal macrophages (MO) stimulated as described with fluorescently labeled zymosan for 20 minutes. (D) Tyrosine phosphorylation of Syk was measured in IFN-γ-primed bone marrow-derived macrophages stimulated for the indicated times with zymosan. Phagocytosis was synchronized by centrifugation of zymosan particles onto the macrophages at 4°C, and then warming the cells to 37°C. (E) Syk expression was detected by intracellular staining and flow cytometry in IFN-γ-primed wild-type and Syk^-/- bone marrow-derived macrophages. (F) Surface expression of Dectin-1, Mac-1 (CD11b), and F4/80 on IFN-γ-primed bone marrow-derived macrophages was measured by flow cytometry.