Necrotic cell death of Mtb-infected macrophages is inhibited by both iron chelation and Fer-1, a well-known ferroptosis inhibitor. C57BL/6 BMDMs were infected with H37Rv Mtb at different MOIs as indicated. (A–C) Macrophages were treated with the iron chelator PIH (1 µM). Cell death and lipid peroxidation were assessed on day 1 p.i. (A) Necrotic cell death measured by Live/Dead staining. (B) LDH release measured in supernatants from macrophage cultures. (C) Lipid peroxidation measured by LAA staining and analyzed by flow cytometry in live (left) and dead cells (right). (D) Representative images of infected macrophage cultures untreated (upper) or treated (bottom) with Fer-1 (10 µm) on day 4 p.i. Dead cells were evaluated by trypan blue staining (bars, 50 µm). (E–G) Macrophage cultures were treated with Fer-1 at the different concentrations indicated. Necrotic cell death was evaluated on day 4 p.i. (E) Necrosis assessed by Live/Dead staining and analyzed by flow cytometry. (F) LDH release measured in supernatants from macrophage cultures. (G) Lipid peroxidation measured by LAA staining and analyzed by flow cytometry in live (upper) and dead cells (bottom). Each data point represents the means ± SEMs of triplicate samples. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Results are representative of three independent experiments performed.