HO-1 protects against heme-induced macrophage cell death. (A) BMM (2 × 10⁵/well) from WT and Hmox1^−/− mice were left untreated (−) or stimulated with increasing concentrations of heme (μM) for 6 hours and supernatants were collected for LDH determination. (B) BMM (10⁶/mL) from WT and Hmox1^−/− mice were pretreated as in panel A and stimulated for 1 hour with heme (30μM) and ROS generation was evaluated by flow cytometry using the probe CM-H2DCFDA (2μM). (C) BMM from WT and Hmox1^−/− mice were pretreated with NAC at 10mM for 1 hour and stimulated with heme (30μM) for 6 hours and supernatants were collected for LDH determination. (D) BMM macrophages (10⁶/mL) from WT and Hmox1^−/− mice were pretreated as in panel C and stimulated for 1 hour with heme (30μM) and ROS generation was determined. (E) BMM from WT and Hmox1^−/− mice were pretreated with deferoxamine (DFX) at 2mM for 2 hours and stimulated with heme (30μM) for 6 hours and supernatants were collected for LDH determination. (F) BMM macrophages (10⁶/mL) from WT and Hmox1^−/− mice were pretreated as in panel E and stimulated for 1 hour with heme (30μM) and ROS generation was determined. Data are representative of 3 independent experiments performed in triplicates and represent mean ± SEM (*P ≤ .05; **P ≤ .001; ***P ≤ .0001).