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

Figure 9

Role of muscarinic receptors in NETosis induced by sera from patients with LAC-associated vasculitis. Neutrophil-enriched suspensions from healthy controls were incubated with HBB (20 nM) for 10 min and then exposed to pooled sera (20%) from healthy controls (PHCs) and patients with LAC-associated vasculitis with (PWN) or without (PWON) GN for 6 h at 37°C. After incubation, cells were stained with Sytox Green and Hoechst 33342, washed, and analyzed by flow cytometry. (a) Representative dot plots and (b) column chart showing the percentage of double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils generated in response to each treatment. Bars represent median values and error bars correspond to IQRs. ; Kruskal–Wallis test. n = 4 independent experiments. (c) Representative images from confocal microscopy showing Sytox Green⁺ Hoechst 33342⁺ netting neutrophils. Magnification: 200x. n = 4 independent experiments.

(a)

(b)

(c)