STAT3-deficient bone marrow granulocytes have an atypical response to G-CSF in vivo. (A-C) Wild-type (WT) or STAT3-deficient (KO) mice were treated with a single dose of G-CSF (250 μg/kg), twice daily doses of G-CSF for 5 days (125 μg/kg every 12 hours), or BSA-containing buffer. Bone marrow cells were collected 24 hours following the single G-CSF dose or 3 hours following the last G-CSF dose in the 5-day treatment regimen. Cells were stained with anti–Gr-1 and anti-CD11b antibodies conjugated to FITC and PE, respectively. (A) Samples were analyzed by flow cytometry, and the percentage of total Gr-1⁺/CD11b⁺ cells (top right quadrant), Gr-1^lo/CD11b⁺ cells (green), and Gr-1^hi/CD11b⁺ cells (blue) is indicated. Data are representative of 3 independent experiments. (B) Absolute levels of Gr-1^lo/CD11b⁺ cells (green), Gr-1^hi/CD11b⁺ cells (blue), or total Gr-1⁺/CD11b⁺ cells (gray) are shown (n = 4 for WT and KO for each condition). Error bars represent SEM. (C) Mononuclear cell counts were determined for each femur (n = 5 for WT and KO for each condition). Error bars represent SEM. (D) The Gr-1^lo/CD11b⁺ and Gr-1^hi/CD11b⁺ populations were isolated from wild-type and STAT3-deficient bone marrow samples by FACS and used for cytospin preparations. Original magnification of the cytospins was × 400. Photos were taken using a Nikon Microphot-FX microscope (Nikon, Garden City, NJ) using a PlanApo 40×/0.95 numerical aperture objective and equipped with a 3-chip charged coupled device (CCD) color video camera (model DXC990; Sony, Tokyo, Japan). Digital images were captured using Optimas Image Analysis software (Media Cybernetics, Silver Spring, MD). Data are representative of 2 independent experiments. (E) Bone marrow Gr-1^lo/CD11b⁺ cells from wild-type and STAT3-deficient mice were isolated by FACS and grown in G-CSF for 3 days ex vivo. Cytospin preparations of the differentiated cells are shown; original magnification was × 400, and images were captured as described for panel D. Data are representative of 2 independent experiments. (F) Forward scatter (FSC) and side scatter (SSC) analysis of Gr-1^lo/CD11b⁺ (black) and Gr-1^hi/CD11b⁺ (purple) populations from bone marrow of WT and KO mice is shown. Data are representative of 5 independent experiments.