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Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification <t>of</t> <t>TLR7/8</t> and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) <t>CL075</t> <t>(1μg/mL),</t> (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.
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Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification <t>of</t> <t>TLR7/8</t> and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) <t>CL075</t> <t>(1μg/mL),</t> (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.
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Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification <t>of</t> <t>TLR7/8</t> and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) <t>CL075</t> <t>(1μg/mL),</t> (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.
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Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification <t>of</t> <t>TLR7/8</t> and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) <t>CL075</t> <t>(1μg/mL),</t> (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.
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Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification of TLR7/8 and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) CL075 (1μg/mL), (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.

Journal: Frontiers in Immunology

Article Title: De novo COVID-19-associated insulin resistance drives dysregulated neutrophil extracellular trap formation (NETosis) four months after infection

doi: 10.3389/fimmu.2026.1787799

Figure Lengend Snippet: Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification of TLR7/8 and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) CL075 (1μg/mL), (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.

Article Snippet: For NETosis, 1 x 10 6 cells were incubated for 30 minutes at 37 °C and 5% CO2 under basal conditions or using IL-6 (20ng/mL) and TNFα (2ng/mL), or TLR7/8 agonists (CL075 (1μg/mL), ImiQ (2μg/mL), and R848 (2μg/mL), ssRNA40 (1μg/mL) and ssRNA 41(1μg/mL) (Catalog No.tlrl-kit3hw3, InvivoGen).

Techniques: Flow Cytometry, Staining, Expressing, Fluorescence