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. 2019 Sep 12;10(9):678. doi: 10.1038/s41419-019-1909-2

Fig. 3. CSE induces more NETs in PMNs of COPD.

Fig. 3

a PMNs of healthy controls and COPD patients were stimulated with 0.3% cigarette smoke extract (CSE) for 4 h at 37 °C. Then neutrophils were fixed with 4% paraformaldehyde. The extracellular DNA was stained with PI (NETs: white arrows). Original magnification, ×400. Scale bars = 50 µm. b Concentrations of extracellular DNA in supernatants of CSE-induced PMNs and PMNs without stimulation (Medium) were investigated by PicoGreen fluorescence quantitative assay. Data are expressed as medians (n = 10). The comparisons were determined by Kruskal–Wallis one-way ANOVA on ranks. *P < 0.05, ***P < 0.001; NS not significant. c PMNs of patients with COPD were stimulated with 0.3% CSE for 4 h at 37 °C, and were fixed with 4% paraformaldehyde. Then components of NETs were stained with NE, MPO or CITH3 (green), and extracellular DNA stained with PI (red) (NETs: white arrows). Original magnification, ×400. Scale bars = 50 µm. d PMNs of patients with COPD were stimulated with 0.3% CSE for 4 h at 37 °C. Concentrations of extracellular DNA in supernatants of PMNs stimulated by CSE and PMNs without stimulation (Medium) were detected by PicoGreen fluorescence quantitative assay. Quantification of NET-associated NE and MPO were detected using the NETosis Assay kit and MPO ELISA kit. Data are expressed as medians (n = 10). The comparisons were determined by Mann–Whitney test. ***P < 0.001