Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum
Fig 8
Gating strategy for analyzing C3 and C5b9 deposition by flow cytometry.
Thresholds and gating on bacteria population on FSC-SSC plots was set up and verified by analyzing signals of 0.2 μm filtered buffer. Even the filtered buffers contained low amount of particles in the size range of bacteria (A) causing low level of inhomogeneity in the bacterial gate. A representative C3 staining of 2% NHS treated ATCC 23505 cells (filled grey histogram untreated cells, dashed line wild type cells, solid black line ecotin KO cells) is shown on panel B. Fluorescence intensity was measured on 20,000 cells captured inside the bacteria gate and C3+ marker was set on the negative control untreated sample. Because of the biasing effect of the few non-bacterial particles in the bacteria gate, the statistical analysis (MFI) of C3-deposition was conducted on the cells under the C3+ marker. Control samples had no event in this region (B). C5b9/PI positive cells were determined by double staining of the cells after treatment. 20,000 cells in the bacteria gate were analyzed, quadrants were set on the untreated, labeled samples. Sample preparation alone induces some cell death (PI positivity) in the samples. Percentage of C5b-9/PI positive cells inside bacteria gate (upper right quadrant) was used for data analysis (C).