S. aureus killing assay

WT LAC and its isogenic mutant strains (10⁹ cfu/ml) were incubated with equal amount of serum from WT mice for 15 min. Neutrophils (5 x 10⁶) enriched from the bone marrow of WT mice by Neutrophil Isolation Kit (Miltenyi Biotec) were cultured with serum-treated S. aureus at a moi of 5 for 15 min, washed with RPMI medium and then treated with recombinant TNF-α (1 μg/ml) for 1 h. After centrifugation, the supernatant and neutrophils were diluted with 0.1% TritonX-100 in PBS and the numbers of extracellular bacteria in supernatant and intracellular bacteria in neutrophils were assessed by serial plating on MSA plates. In some experiments, neutrophils cultured with YFP-A aureus were stained with APC-conjugated anti-Ly6G antibody, fixed with 4% paraformaldehyde, treated with 0.1% saponin, incubated with rabbit anti-Rab5 antibody, stained with DAPI and Alexa Fluor 555-conjugated anti-rabbit IgG or PE-labeled anti-LAMP-1 antibody, and viewed with confocal microscope A1 (Nikon).