G-CSF-induced intracellular ROS production. Ba/F3 cells stably transfected with the wild-type G-CSF receptor were treated with variable G-CSF concentrations or for variable time periods. ROS production was measured in cells labeled with DHR. ROS levels induced by G-CSF normalized to that of untreated cells were determined by monitoring the increased fluorescence in the cells. (A) Dose-dependent increase in intracellular ROS production (mean ± SD). (B) Time course for increased intracellular ROS production (mean ± SD). (C) G-CSF-induced neutrophil differentiation. (Left) 32DGR cells grown in medium with IL-3 appear as myeloblasts. (Right) 32GR cells grown in medium with 100 ng/mL G-CSF for 10 days display a population of granulocytes (Giemsa staining; × 200 magnification). Photographs were obtained using a Nikon Microphot-FX microscope (Nikon, Tokyo, Japan) and a Nikon plan/apo 20×/0.75 numeric aperture objective lens. Images were captured using a Sony 3CCD color video camera, model DXC-990 (Sony, Tokyo, Japan) and were processed using Acquisitions software (Media Cybernetics, Silver Spring, MD). (D) G-CSF-induced intracellular ROS production in 32DGR cells. Purple indicates unstimulated cells; green, G-CSF for 30 minutes; red, 100 μM H₂O₂ for 30 minutes.