Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum
Fig 10
Intense NHS-driven cell-lysis is detected by DNA-content of cell supernatants.
We examined MAC formation both by C5b-9 specific antibodies and by propidium iodide (PI) to identify bacteria killed by MAC-driven cell lysis. In several samples, where cells were treated with above 4% NHS, the C5b-9 MFI values of the C5b-9/PI positive cells topped or even decreased (Fig 9C and 9F) indicating some artefacts. Here we show that the supernatants of these cells contain large amounts of DNA suggesting that the highest-labeled C5b-9/PI positive population has been disintegrated. Supernatants obtained during the preparation for flow cytometry experiments were applied onto agarose gel and DNA was detected with SYBR Green. No significant amounts of DNA leaked from wild type ATCC 23505 cells upon incubation with serum up to 16% NHS (A), while its ecotin KO derivative already lysed in as low as 1% NHS (B). The supernatant of ATCC 12014 cells contain detectable (C), while that of its ecotin KO derivative significant amount (D) of DNA upon treatment with 8% or 16% NHS. The % values indicate the NHS concentration the cells were treated with.