Phagocytosis and Bacteria Killing Assay

BM-derived macrophages were seeded on coverslips in 24-well plates. Escherichia coli (BL21) (1 × 10⁷) carrying an EGFP-expressing plasmid were added to BMDMs at a 10:1 ratio (bacteria:BMDMs), followed by incubation at 37°C for 2 h. Free E. coli were removed by washing and the remaining cells were stained with biotin-anti-F4/80 antibody (eBioscience, San Diego, CA, USA), followed by Cy3-avidin (eBioscience). Samples were visualized under a fluorescence microscope (BX51, Olympus, Tokyo, Japan) or analyzed by flow cytometry, and the average number of engulfed EGFP⁺ E. coli per macrophage was calculated. To determine bacteria killing by macrophages, macrophages that had engulfed EGFP⁺ E. coli were incubated for a further 6 h. Cells were then lysed, diluted, and plated on ampicillin-containing agar plates. Bacterial colonies were counted and compared after overnight culture.