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. 2016 Dec 1;7:557. doi: 10.3389/fimmu.2016.00557

Figure 1.

Figure 1

Diamond nanoparticles induce plasma membrane damage and NET formation in human leukocytes. (A) Assessment of DNA exposure in neutrophils (PMN) and peripheral blood mononuclear cells (PBMC). Cells without stimulus (wo) or incubated with nanodiamonds (10 nm) or microdiamonds (1000 nm) were measured using SYTOX Green. (B) Exposure of DNA by neutrophils was measured in response to phorbol 12-myristate 13-acetate (PMA) to nanodiamonds (10 nm) or microdiamonds (1000 nm) and without stimulus (wo) using SYTOX Green. (C) Dose-dependent increase of DNA exposure in PMN without stimulus (wo) or incubated with nanodiamonds (10 nm) or microdiamonds (1000 nm) by SYTOX Green after 150 min. (D) Microscopic analysis of PMN incubated with nanodiamonds (10 nm) or microdiamonds (1000 nm) and stained for DNA (Hoechst33342), neutrophil elastase (NE), or citrullinated histone H3 (citH3). Diamonds are visible in differential interference contrast (DIC) images. Cy5 fluorescence was artificially colored green. (E) Microscopic analysis of PBMC incubated with nanodiamonds (10 nm) or microdiamonds (1000 nm) and stained for DNA (Hoechst33342) and CD45-FITC. Diamonds are visible in DIC and provide strong background fluorescence in FITC. (F) Quantification of extracellular DNA and citH3 in human PMN without stimulus (wo) or incubated with nanodiamonds (10 nm) or microdiamonds (1000 nm) after 240 min. Significances below 0.001 are depicted. (G) Microscopic analysis of PMN incubated with nanodiamonds and treated with the ROS scavenger N-acetyl cysteine stained for DNA (Hoechst33342), NE, or citH3. Diamonds are visible in DIC. (H) In silico quantification of microscopic pictures regarding the number and area of DNA (Hoechst33342)-stained NET structures (AggNET) formed by PMN incubated with nanodiamonds in the absence or presence of the ROS scavenger N-acetyl cysteine. Each dot represents one analyzed field of view (FOV). (I) Microscopic analysis of NET formation in response to nanodiamonds induced necrosis of CFSE-labeled PBMC stained for DNA (Hoechst33342), NE, or citH3. Diamonds are visible in DIC. Data of one representative experiment reflecting the result of three independent experiments are shown as medians with interquartile ranges of triplicates. Two-way ANOVA (A–C), one-way ANOVA (F), Kruskal–Wallis one-way analysis of variance (H), and Mann–Whitney U test (I) were used to evaluate differences among means; **p < 0.05, ***p < 0.001, and relative fluorescence units (RFU) field of view (FOV).