American Society of Hematology

Figure 3.

$Impaired NET formation by neutrophils derived from a patient with a mutation in ELANE. (A) Protease release and proteolytic activity of neutrophils and eosinophils upon stimulation with CytoB/fMLF (5 μg/mL; 1 μM) and CytoB/PMA (5 μg/mL; 100 ng/mL) were determined by DQ-green BSA. A Triton X-100 lysate was used as 100% (mean + standard deviation, n = 3). (B) Representative cytospins of isolated granulocyte fractions from controls and patient (original magnification ×400; May-Grünwald/Giemsa stain). These cell suspensions were used in panel F. ∗eosinophil, ¥neutrophil, xlymphocyte. (C) Protease release and proteolytic activity by control (n = 5) and ELANE patient (n = 3) granulocytes (mean + standard deviation). (D-F) To induce ET formation, cells were stimulated with PMA (100 ng/mL) for 4 hours, and (D) DNA release was measured in real-time by Sytox Green Nucleic Acid Stain (mean + standard deviation, n = 4 for controls, n = 3 for ELANE patient) and (E) ETs were visualized by staining for neutrophil elastase (NE, green, 488), DNA (Hoechst, blue, 405), and myeloperoxidase (MPO, magenta, 633). Images were acquired using a Leica SP8 confocal microscope, original magnification ×400. Scale bar = 50 μm. Results are representative of 3 independent experiments. (F) NETosis dynamics upon PMA stimulation were assessed by live cell imaging using the permeable DNA dye DRAQ5 (cyan) and the impermeable DNA dye Sytox Green (magenta). Asterisk indicates an eosinophil. Time is displayed in hours, scale bar = 20 μm. Refer to supplemental Video 8. Results are representative of 2 independent experiments. (A) Statistics were performed by unpaired t test; ∗∗P < .01; ∗∗∗P < .001.$

Impaired NET formation by neutrophils derived from a patient with a mutation in ELANE. (A) Protease release and proteolytic activity of neutrophils and eosinophils upon stimulation with CytoB/fMLF (5 μg/mL; 1 μM) and CytoB/PMA (5 μg/mL; 100 ng/mL) were determined by DQ-green BSA. A Triton X-100 lysate was used as 100% (mean + standard deviation, n = 3). (B) Representative cytospins of isolated granulocyte fractions from controls and patient (original magnification ×400; May-Grünwald/Giemsa stain). These cell suspensions were used in panel F. ∗eosinophil, ^¥neutrophil, ^xlymphocyte. (C) Protease release and proteolytic activity by control (n = 5) and ELANE patient (n = 3) granulocytes (mean + standard deviation). (D-F) To induce ET formation, cells were stimulated with PMA (100 ng/mL) for 4 hours, and (D) DNA release was measured in real-time by Sytox Green Nucleic Acid Stain (mean + standard deviation, n = 4 for controls, n = 3 for ELANE patient) and (E) ETs were visualized by staining for neutrophil elastase (NE, green, 488), DNA (Hoechst, blue, 405), and myeloperoxidase (MPO, magenta, 633). Images were acquired using a Leica SP8 confocal microscope, original magnification ×400. Scale bar = 50 μm. Results are representative of 3 independent experiments. (F) NETosis dynamics upon PMA stimulation were assessed by live cell imaging using the permeable DNA dye DRAQ5 (cyan) and the impermeable DNA dye Sytox Green (magenta). Asterisk indicates an eosinophil. Time is displayed in hours, scale bar = 20 μm. Refer to supplemental Video 8. Results are representative of 2 independent experiments. (A) Statistics were performed by unpaired t test; ∗∗P < .01; ∗∗∗P < .001.

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