R phycoerythrin

Experiments were performed based on methods described previously (133 (link)). Briefly, mid log phase bacteria grown in the presence or absence of Sia were washed in HBSS containing Ca/Mg and then treated with 10% NHS for 5 min at 37°C and 200 rpm. For IgG and IgM deposition, NHS was heat inactivated by incubation at 56°C for 30 min and 200 rpm shaking. Heat-inactivated NHS-treated bacteria were incubated with R-phycoerythrin- or FITC-conjugated antibodies against human IgG and IgM (both Sigma-Aldrich), respectively. For complement component, C3c was probed with FITC-conjugated anti-human C3c (Bio-Rad). For factor H 6/7 binding, the bacteria were incubated with the purified FH6/7 (mouse IgG) supernatant (134 (link)) for 30 min and visualized using allophycocyanin (APC)-conjugated anti-mouse IgG. Siglec-9−Fc binding was determined by incubating the bacteria with 300 ng of purified protein for 2 h at 37°C. The visualization was done using Alexa Fluor 488-conjugated secondary antibody against human Fc. All the data were collected using BD FACSCalibur flow cytometer, and the data were analyzed using FlowJo software.

Penicillin-streptomycin solution, trypsin-EDTA solution, Dulbecco’s modified Eagle’s medium, RPMI 1640 medium, and 1% antibiotic-antimycotic solution were obtained from Life Technologies/Gibco (Grand Island, NY, USA). AgNO₃, fetal bovine serum, and the in vitro toxicology assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Graphite (Gt) powder, NaOH, KMnO₄, NaNO₃, anhydrous ethanol, 98% H₂SO₄, 36% HCl, 30% H₂O₂ aqueous solution, silver nitrate, R-phycoerythrin, and all other chemicals were purchased from Sigma-Aldrich unless otherwise stated.