Stat5a/b-null neutrophils have normal functions but are less responsive to GM-CSF–permitted survival. (A) Phosphoflow cytometry for STAT1, STAT3, and STAT5A in neutrophils derived from bone marrow of Stat5f/f and Stat5f/f:Mx1-Cre mice. Cells were stimulated with GM-CSF for 15 minutes after 3 hours of starvation. One representative experiment is displayed (n = 5 experiments). Control experiments are shown in supplemental Figure 2. (B) Migration assay by the induction of peritoneal exudates induced by the intraperitoneal injection of sodium TGA. At 4 hours after injection, peritoneal neutrophils from Stat5f/f and Stat5f/f:Mx1-Cre mice were counted. Data are mean ± SD (n = 18 mice per group for TGA, n = 4 mice per group for phosphate-buffered saline). (C) Phagocytosis assay with bone marrow neutrophils from Stat5f/f and Stat5f/f:Mx1-Cre mice. Flow cytometry determined the level of phagocytosis of Texas Red–conjugated opsonizing zymosan in Gr1+ neutrophils. Data are mean ± SD; n = 3 mice per group. One representative experiment is shown. (D) Oxidative burst assay. Superoxide production was determined in bone marrow mature neutrophils from Stat5f/f and Stat5f/f:Mx1-Cre mice. Cells were preincubated with hydroethidine and then activated by phorbol myristate acetate. Results (mean ± SD) are given as percentage to the control; n = 3 mice per group. (E) Survival assay. Bone marrow neutrophils from Stat5f/f and Stat5f/f:Mx1-Cre mice were cultured as indicated for 36 hours. Data are means ± SD (n = 3 mice per group). *P = .02.
