<p>(<b>A</b>) and (<b>B</b>) Spores were incubated with purified human C3b, CFH and CFI. C3 fragments deposited on the spore surface were detected using anti-C3 polyclonal antibodies (<b>A</b>). The image shown is representative of at least three independent experiments. The ratio of iC3b/C3b was determined by quantifying the density of the corresponding bands in western blots using Image J (<b>B</b>). The β chain represents C3b + iC3b, and the α” chain represents iC3b. Results were combined from three independent experiments. (<b>C</b>) and (<b>D</b>) Rate of iC3b deposition on spores. Spores were incubated with 10% NHS for the indicated time and subjected to flow cytometry analysis using iC3b-specific antibody. The results shown are mean fluorescence intensity normalized to that at 10 minutes, respectively, and combined from at least three independent experiments (<b>C</b>). The rate of iC3b deposition (ΔiC3b/Δt) was calculated by linear regression analysis of the normalized data (GraphPad Prism 6) (<b>D</b>). (<b>E</b>) Determination of C3a concentration. GVB<sup>0</sup> buffer containing 20% NHS (no antibody), 20% NHS pre-treated with the CFH functional blocking antibody OX24 (240 nM final conc.) or mouse IgG1 control (240 nM) was incubated with no spore, 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA spores at 37°C for 30 min and centrifuged to remove the spores. C3a concentrations in the supernatants were determined using the Human C3a ELISA kit (BD OptEIA<sup>™</sup>) and normalized to the respective no spore control. Data was combined from four experiments, each with duplicate wells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; <i>t</i> test.</p
