Fig. 1. Responsiveness of human neutrophils to 15 min stimulation with IL-4, IL-13 and IFN-γ, measured by phosphorylation of STAT6 and STAT1 using flow cytometry.

(A) Gating strategy for neutrophils, monocytes and lymphocytes. Neutrophils were defined as CD11b^high CD16^high cells. Monocytes and lymphocytes were gated based on forward and side scatter characteristics and CD16 + cells were excluded from cells in lymphocyte gate. Doublets were excluded from all three populations (not shown). (B) Stimulation of human whole blood was performed with indicated amounts of IL-4 or IL-13 and phosphorylation of STAT6 was determined in CD11b^highCD16^high cells. Representative histograms of fluorescence intensity are shown. (C) Geometrical means ± SEM of pSTAT6 fluorescence of six donors after stimulation with IL-4 or IL-13. The curves of both stimulations are fitted using non-linear regression 3-parameter fit (goodness of fit was evaluated with R square and the values were for IL-4 and IL-13 0.8604 and 0.8961, respectively) and EC₅₀ values are indicated based on the fitted curves. The IL-4 10 ng/ml datapoint was lost for one donor. (D) Geometrical means of pSTAT1 (pY701) fluorescence of six donors after whole blood was either left untreated or stimulated with 100 ng/ml of IFN-γ for 15 min, *) p = 0,0313; Wilcoxon matched-pairs signed rank test.