Bacterial Binding of Factor H Assay

Factor H (FH) binding to bacteria was performed using flow cytometry as described previously [16 (link)]. Briefly, bacteria (Ng F62 ΔlgtD) were harvested from chocolate agar plates and grown in liquid media that contained the specified concentration of the CMP-NulO as described above. Bacteria were then washed with Hanks Balanced Salt Solution (HBSS) containing 1mM Ca²⁺ and 1 mM Mg²⁺ (HBSS⁺⁺) and incubated with FH purified from human plasma (Complement Technology, Inc.; concentration specified for each experiment). Bound FH was detected using either anti-FH mAb (Quidel, catalog no. A254 (mAb 90X)) or affinity-isolated polyclonal goat anti-human FH, followed by FITC conjugated anti-mouse IgG or anti-goat IgG, respectively (Sigma); both Abs had similar performance characteristics. All reaction mixtures were carried out in HBSS⁺⁺/1% BSA in a final volume of 50 μl. Flow cytometry was performed using a FACSCalibur instrument (Becton Dickinson) and data were analyzed using FlowJo (version 7.2.5; Tree Star, Inc.).