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

Figure 8

Role of muscarinic receptors in NETosis induced by a cocaine-levamisole mixture. Neutrophil-enriched suspensions were incubated with 20 nM HBB for 10 min and then exposed to cocaine (20 μM), levamisole (40 nM), and the cocaine-levamisole mixture (20 µM/40 nM) and PMA (20 nM) for 6 h at 37°C. After incubation, cells were stained with Sytox Green and Hoechst 33342, washed, and analyzed by flow cytometry. (a-b) Representative dot plots and column chart showing the percentages of double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils induced by each treatment. Bars show median values and error bars correspond to IQRs. ; Kruskal–Wallis test. n = 4 independent experiments. (c) Immunofluorescence analysis by confocal microscopy confirming the inhibition of NET formation by HBB. Magnification: 200x. n = 4 independent experiments.

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