ROS are essential to the cytotoxic effect of heme. (A-C) BMM (2 × 10⁵/well) from Tlr4^−/− mice were left untreated (−) or pretreated for 1 hour with NAC at 10mM or pretreated for 2 hours with deferoxamine (DFX) at 2mM, stimulated for 6 hours with heme at 30μM, and/or TNF at 0.5 ng/mL (+) and supernatants were collected for LDH determination. (B-D) Peritoneal macrophages (10⁶/mL) were pretreated as in (A-C) and stimulated for 1 hour with heme (30μM) and ROS generation was evaluated by flow cytometry using the probe CM-H2DCFDA (2μM). (E) Macrophages from C57Bl/6 mice were left untreated or stimulated with heme at 30μM, and/or TNF at 0.5 ng/mL in the presence or absence of FCS at 10%. After 6 hours supernatants were collected for LDH determination. (F) Peritoneal macrophages (10⁶/mL) were stimulated for 1 hour with heme (30μM) in the absence or presence of FCS at 10% and ROS generation was evaluated by flow cytometry using the probe CM-H2DCFDA (2μM). Data are representative of 3 independent experiments performed in triplicates and represent mean ± SEM (*P ≤ 0,05; ***P ≤ .0001).