Heme stimulates NET formation in neutrophils in vitro. (A) Representative images of plasma of TNF-α–stimulated SCD mice and SA mice. (B) Quantification of plasma heme concentrations of TNF-α–stimulated SA mice (black circle, n = 8), TNF-α–stimulated SCD mice (white circle, n = 5), and PBS-treated SCD mice (gray circle, n = 6; mean ± SEM, ***P < .001). (C) Quantification of NETs released by TNF-α–primed neutrophils stimulated with 100 nM phorbol myristate acetate (PMA) (white bar), 10 μM heme (blue bar), and 20 μM heme (red bar). The shaded bars represent DMSO treatment of each condition. Results are the average values of 3 independent experiments (mean ± SEM, *P < .05). (D) Representative immunofluorescence images of NETs released by TNF-α–primed neutrophils stimulated with (i) 100 nM PMA, (ii) 20 μM heme, (iii) DMSO, and (iv) 20 μM heme; the slide was then treated with DNase I. (E) Representative immunofluorescence images of DNA (green/SYTO13), H3Cit (red/Alexa Fluor 568, i,ii), or NE (red/Alexa Fluor 568, iii,iv) of NETs generated by TNF-α–primed wild-type neutrophils that were stimulated with 20 μM heme. Scale bar, 10 μm.