NETosis Secondary to the Use of Levamisole-Adulterated Cocaine: A Likely Underlying Mechanism of Vasculopathy

Figure 4

Induction of NETosis in neutrophil cultures exposed to sera from patients with LAC-associated vasculitis. Neutrophil-enriched suspensions from healthy controls were exposed to pooled sera (20%) from healthy controls (PHCs) and patients with LAC-associated vasculitis with GN (PWN) or without GN (PWON) for 6 h at 37°C. Staining with Sytox Green, Hoechst 33342, and anti-MPO-PE and anti-NE-Alexa fluor 647 mAbs was used to evaluate extrusion of neutrophil DNA and granular MPO. (a-b) Representative dot plots and column chart showing the percentages of double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils generated in response to each treatment. (c-d) Representative dot plots and column chart showing the percentages of double positive MPO ⁺ Hoechst 33342⁺ netting neutrophils generated in response to each treatment. Bar represents median values and error bars correspond to IQRs. ; Kruskal–Wallis test. n = 4 independent experiments. (e) Immunofluorescence analysis by confocal microscopy confirming NET formation. Magnification: 200x. n = 4 independent experiments.

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