Phagocytosis Assays

The macrophages (i.e., RAW 264.7 cells, HMDMs) were seeded in 24-well plates (2 × 10⁵ cells/well) in antibiotics-free medium the day before infection, and then infected with GFP-expressing PAO1 and PAO1 sodB P. aeruginosa strains, at a MOI of 25. Phagocytosis was carried out by incubation of the infected macrophages for 30 min or 60 min at 37°C in 5% CO₂. Afterward, the cells were gently washed two or three times with PBS, enzymatically detached, and analyzed by flow cytometry (BD FACSCalibur, France). Phagocytosis was evaluated as the fraction of GFP⁺ cells in the bulk population. The data were analyzed using the CellQuest software, and the images were processed with FlowJo.