The ability of Dectin-1 to activate Syk and ROS production is a transient property of a macrophage. (A) IFN-γ-primed bone marrow-derived macrophages were stimulated for 20 minutes with TRITC-labeled zymosan (first zymosan pulse), washed, and then stimulated with a second round of R-PE-labeled zymosan for an additional 207 minutes (second zymosan pulse). Populations of cells internalizing zymosan in the first, second, and both periods were identified by flow cytometry. (B) Syk activation was measured in cells using the gates indicated in panel A. (C) RAW264.7 cells expressing SBP-tagged Dectin-1 were stimulated with TRITC-zymosan for 1 hour, and ROS production was measured by flow cytometry usingAPF. Cells internalizing zymosan but not activating ROS production (ROS^-) and cells in which ROS production was activated (ROS⁺) were sorted and expanded for 5 days in culture. (D) The sorted cells from panel C were restimulated with TRITC-zymosan and ROS production was again measured by flow cytometry. (E) Bone marrow-derived macrophages were treated overnight with IFN-γ (25 U/mL), IL-4 (20 ng/mL), or PAM₃CSK₄ (100 ng/mL) as indicated, and zymosan-induced activation of Syk was measured by flow cytometry. Data are gated on cells that had eaten zymosan.