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

Figure 2

Induction of NETosis in neutrophil cultures exposed to cocaine, levamisole, and cocaine-levamisole mixture. Neutrophil-enriched suspensions from healthy controls were exposed to cocaine (40 µM), levamisole (20 nM), cocaine-levamisole mixture (20 µM/40 nM), and PMA (20 nM) for 6 h at 37°C. Staining with Sytox Green, Hoechst 33342, and mAbs anti-MPO-PE and anti-NE-Alexa fluor 647 was used to evaluate extrusion of neutrophil DNA and granular content. (a) Representative dot plots illustrating the strategy for gating double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils. (b-c) Representative dot plots and column chart showing the percentage of netting neutrophils in response to each treatment. Bar represents median values and error bars correspond to IQRs. ; Kruskal–Wallis test. n = 4 independent experiments. (d) Immunofluorescence analysis by confocal microscopy confirming NET formation. Magnification: 200x. n = 3 independent experiments.

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