STAT3 is required to maintain a normal ratio of immature to mature neutrophils in peripheral blood and spleen. Wild-type (WT) and STAT3-deficient (KO) mice were injected at 12-hour intervals with G-CSF (125 μg/kg) or BSA-containing buffer for 5 days. Peripheral blood and spleen samples were collected 3 hours following the last injection and stained with anti–Gr-1 and anti-CD11b antibodies conjugated to FITC and PE, respectively. (A) Blood 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. Because STAT3-deficient mice show variable neutrophilia, animals with similar basal levels of peripheral Gr-1⁺/CD11b⁺ cells were chosen for comparison. Results are representative of at least 2 independent experiments (n = 4 for WT and KO for each condition). (B-C) Absolute levels of Gr-1^lo/CD11b⁺ cells (green), Gr-1^hi/CD11b⁺ cells (blue), or total Gr-1⁺/CD11b⁺ cells (gray) in blood (B) or spleen (C) are shown (n = at least 4 for WT and KO for each condition). (D) Mononuclear cell counts were determined for individual spleens (n = 4 for WT and KO for each condition). (E) Total hematopoietic neutrophil levels were determined per mouse as described in “Materials and methods.” Average neutrophil numbers are shown (n = 4 for WT and KO for each condition). Error bars represent SEM in all panels.