iIFNAR−/− macrophages were grown in 35 mm-diameter dishes at a density of 2 × 106 per dish. Cells were infected with L. monocytogenes at an MOI of 10 for 6 h or treated for 1 h with 100 µM menadione (positive control). N-acetylcysteine (NAC) (1 mM) was added to cell cultures throughout treatment to scavenge ROS. Treated cells were stained with 2.5 µM CellROX DeepRed (Molecular Probes; C10422) for 30 min at 37°C. Cells were then washed twice in FACS buffer, fixed with 3.7% formaldehyde for 15 min, and washed again prior to analysis on a BD LSR-II flow cytometer.