Heme-mediated NET formation in TNF-α–primed neutrophils is dependent on ROS and heme-iron and can be blocked by hemopexin complexation. (A) Quantification of NETs released by TNF-α–primed neutrophils treated with DMSO (white bar), antioxidant NAC alone (10 mM, blue bar), heme alone (20 μM, black bar), heme (20 μM), and NAC (10 mM) (gray bar). Results are the average values of 3 independent experiments (mean ± SEM, **P < .01, ***P < .001). (B) Quantification of NETs released by TNF-α–primed neutrophils treated with free heme (black bar) or Hx complex (gray bar). Results are the average values of 3 independent experiments (mean ± SEM, *P < .05). (C) Quantification of NETs released by TNF-α–primed neutrophils treated with free heme (black bar) or HSA complex (gray bar). Results presented are the average values of 3 independent experiments (mean ± SEM). (D) Quantification of NETs released by TNF-α–primed neutrophils treated with free heme (black bar) or HbA (gray bar). Results are the average values of 4 independent experiments (mean ± SEM, **P < .01). (E) Representative immunofluorescence images of NETs released by TNF-α–primed neutrophils stimulated with 20 μM of (i) heme, (ii) zinc (II) protoporphyrin IX (Zn (II) PPIX), (iii) protoporphyrin (PPIX), and (iv) iron (III) mesoporphyrin IX (Fe (III) Meso). (F) Quantification of NETs released by TNF-α–primed neutrophils treated with DMSO (white bar), heme (black bar), Zn (II) PPIX (red bar), PPIX (blue bar), and Fe (III) Meso (gray bar). Results are the average values of 3 independent experiments (mean ± SEM, *P < .05, ***P < .001).