N acetyl cysteine (nac)

Ascorbic acid (Kobayashi Pharmaceutical, Osaka, Japan) and NAC (LKT Laboratories, Minnesota, USA) were dissolved in saline. Trolox (Tokyo Chemical Industry, Tokyo, Japan) was dissolved in DMSO (Wako Pure Chemical Industries).

Femoral marrows from wild-type mice were flushed and cells were plated at density 1 × 10⁶ cells/mL in culture petri dishes (Becton Dickinson) using Dulbecco's minimal Eagles medium (Invitrogen) supplemented with 10% fetal bovine serum, 10 mM sodium pyruvate, 10 mMl-glutamine, penicillin, and streptomycin (Sigma Aldrich, St. Louis). The cells were differentiated with 20% mouse L929 fibroblast cell line culture supernatant as a source for macrophage-colony stimulating factor. After 4 days of culture, nonadherent cells were removed and adherent cells, bone marrow-derived macrophages (BMDMs), were washed twice with PBS and the medium was replaced daily for 7 days, when the cells were used for the experiments.
BMDMs were subjected to treatments with the following compounds: heme (25 μM; for 4 and 16 h), heme in combination with NAC (1 mM; LKT laboratories, Inc.) 1 h before heme treatment, with PPIX (25 μM, for 4 h; Sigma Aldrich), DFO (100 μM; Sigma Aldrich), allopurinol (1 mM; Sigma Aldrich), apocynin (100 μM; Sigma Aldrich), LPS (100 μg/mL, for 4 h; from L2880 E. coli, serotype 055:B5; Sigma Aldrich), or with polymyxin B sulfate salt (0.1 μg, 1 μg, and 10 μg/mL) (Sigma Aldrich).