Opsonophagocytic killing assay

In the ex vivo whole-blood killing model [15], blood was taken from anesthetized BALB/c mice (female, 6-week-old) by cardiac puncture and collected in a vial containing Heparin (Sigma). PBS-washed USA300 was adjusted to a concentration of 3×10⁵ CFU/ml. Then, 100 μl of S. aureus was mixed with 200 μl of pooled mouse blood containing 10 μg/ml 2H7 or isotype control antibody. In addition, a control experiment was conducted to address the possibility that antibodies mediate bacterial aggregations and result in a drop in apparent CFUs. Blood cells were first lysed in the presence of 0.5% saponin and further incubated with USA300 and antibodies as described above.

Opsonophagocytic killing with human neutrophils was conducted as follows. HL60 cells (ATCC) were cultured, differentiated to granulocytes and washed as described previously [27]. In 5 to 6 days, 80 to 85% of N, N-dimethyl- formamide-induced HL60 cells were differentiated into granulocytes. Guinea pig serum was used as complement source. To remove the cross-reactive antibodies, serum was incubated with S. aureus USA300 (4°C for 30 min), centrifuged and filter-sterilized. The assay was performed in a 96-well plate containing 1×10⁴ CFU USA300, 1×10⁵ viable differentiated-HL60 cells, 4% complement serum and 10 μg/ml 2H7 or isotype control antibody. The mixed samples were incubated at 37°C with shaking. At 0 and 60 min, 10 μl aliquots were removed and diluted with 0.5% saponin-PBS. Then, the samples were sonicated briefly to disrupt bacterial aggregates and plated on TSA to determine the viable CFU numbers. The relative killing was calculated as the percent difference in CFU between samples at 0 min and 60 min.