Enhanced erythrophagocytosis induces ferroptosis in macrophages in vitro. Bone marrow was harvested from wild-type C57BL/6 mice and cultured in the presence of macrophage colony-stimulating factor (20 ng/mL) for defined times to determine when the cell population uniformly expressed VCAM-1 and F4/80. (A) Representative dot plots (the percentages of double-positive cells are indicated), (B) frequency of VCAM-1⁺, F4/80^hi macrophages, and (C) overall VCAM-1 expression are shown. BMDMs and J774 cells were plated 24 hours prior to the experiments and then incubated with PBS, fresh RBCs, or IgG-opsonized RBCs, which were labeled with CellTrace Far Red. The amount of erythrophagocytosis in (D) BMDMs and (E) J774 cells was quantified by flow cytometry. J774 cells (F-G) and BMDMs (H-I) were treated with vehicle control or 100 μM Fer-1 and exposed to IgG-opsonized RBCs; they were then analyzed for ROS (F,H) and lipid peroxidation (G,I). Twenty-four-hour exposure to erastin (20 μM), RSL3 (1 μM), and IKE (20 μM), which are potent ferroptosis inducers, induced BMDM (J) and J774 cell loss (K). BMDM cell viability was quantified following incubation with IgG-opsonized RBCs or PBS, and with vehicle control or 100 μM Fer-1 for 6 hours; enhanced erythrophagocytosis induced substantial cell death, which was ameliorated by Fer-1 (L). Data are representative of 3 independent experiments. *P < .05; ***P < .001; ****P < .0001; ANOVA with the Dunnett multiple comparisons test.