Measuring Bacterial Factor H Binding

Factor H (FH) binding to bacteria was performed using flow cytometry as described previously (44 (link)). Briefly, N. gonorrhoeae F62 ΔlgtD harvested from chocolate agar plates was grown in liquid media that contained the specified concentration of the CMP-NulO. Bacteria were then washed with Hanks Balanced Salt Solution (HBSS) containing 1 mM CaCl₂ and 1 mM MgCl₂ (HBSS⁺⁺) and incubated with 20 µg/ml of FH purified from human plasma (Complement Technology, Inc.). Bound FH was detected using an anti-FH mAb (Quidel; clone 90X), followed by FITC conjugated anti-mouse IgG (Sigma); both Abs had similar performance characteristics. All reaction mixtures were carried out in HBSS⁺⁺/1% (w/v) BSA in a final volume of 50 µl.