FIGURE 5.

Effector function of neutrophils derived in vivo from HoxB8-conditional progenitors. Seven days after CD45.1 mice received transplant of wild-type HoxB8-conditional progenitors, blood samples were subjected to assays of neutrophil function using flow cytometry. CD45.2⁺Ly6G⁺ donor-derived neutrophils were distinguished from CD45.1⁺Ly6G⁺ host-derived neutrophils for comparative analyses. (A) Host and donor neutrophil phagocytosis of pHrodo-Green conjugated S. aureus, measured after 60 min exposure in the absence or presence of 10 μg/ml cytochalasin D (CD). Data were analyzed using two-way ANOVA with Bonferroni post-hoc test for multiple comparisons. *p < 0.05, compared to 0 min #p < 0.05, compared to 60 min. (B) Host and donor neutrophil intracellular killing of S. aureus, as measured by the fraction of neutrophils remaining GFP⁺ 45 min after allowing initial phagocytosis of strain USA300-sGFP. The killing efficiency was calculated by comparing the end point fraction of GFP⁺ neutrophils to the starting point (0 min). Data were analyzed using two-way ANOVA with Bonferroni post-hoc test for multiple comparisons. *p < 0.05, compared to 0 min #p < 0.05, as indicated. Killing efficiency data were analyzed by an unpaired Student’s t-test. (C) Quantification of stimulated ROS generation by host and donor neutrophils, expressed as the mean fluorescence intensity of DHR123. Blood samples were stimulated with either heat-killed S. aureus (HKSA) or 100 ng/ml PMA, in the absence or presence of 10 μM DPI. Data were analyzed using two-way ANOVA with Bonferroni post-hoc test for multiple comparisons. *p < 0.05, compared to unstimulated. #p < 0.05, compared to HKSA or PMA alone.