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. 2021 Dec 18;10(12):3581. doi: 10.3390/cells10123581

Figure 2.

Figure 2

Pneumolysin triggers the formation of neutrophil extracellular traps (NETs). (A,B) NET formation was quantified using the cell-impermeant nucleic acid dye, SYTOX Green, in human neutrophils treated with PLY (100 or 500 ng/mL). (A) NET production (SYTOX Green fluorescence) was monitored over a period of 4 h (representative graph), (B) Bars represent the area under the curve (AUC) of Sytox Green fluorescence for Ctr and PLY-treated cells calculated from the kinetic curves. n = 12, * p < 0.05, ** p < 0.01, one-way ANOVA. (C) NET production in neutrophils pre-treated with U0126 (25 μM) and then challenged with PLY (100 ng/mL). AUC for each condition was calculated from the kinetic curves (Sytox Green assay). Each line represents neutrophils from the same donor differentially treated. n = 4. * p < 0.05, paired t-test. (D) Neutrophils were treated with PLY, fixed, and stained with the nuclear stain DAPI. Depicted are representative pseudocolor images. Red represents intact nuclei. White staining indicates extracellular DNA. Arrows indicate extracellular DNA structures. Pictures were taken at 40x magnification. Scale bar: 50 μm. n = 3. (E) Elastase activity was measured in isolated NETs from PLY-treated or control neutrophils. n = 7–9. * p < 0.05, *** p < 0.001, one-way ANOVA.