Human neutrophil cell surface Siglec-9 interacts with sialic acids presented in trans. (A) Neutrophils bind to immobilized α2-3–linked Sias. Wells were coated with polyacrylamide arrays carrying multiple copies of Siaα2-3Galβ1-4GlcNAc or Galβ1-4GlcNAc (100 μL of 1 mg/mL stock diluted 1:100; GlycoTech, Gaithersburg, MD). Fluorescently labeled neutrophils were added, allowed to adhere, and unbound cells then washed away. The percentage of adherent neutrophils was determined by dividing the final FI by initial FI and multiplying by 100. (B) Validation of IgG anti–Siglec-9 Sia binding site (BSAb) and Sia nonbinding site (NBSAb) antibodies as functional reagents; 96-well plates were coated with 100 μL protein A in carbonate buffer (2 μg/mL) overnight at 4°C. Recombinant soluble Siglec-9-Fc chimera was then added in 100 μL enzyme-linked immunosorbent assay (ELISA) buffer (2 μg/mL) overnight at 4°C. The wells were washed, pretreated with BSAb, NBSAb, or PBS for 30 minutes at room temperature, and then washed 3 times with ELISA buffer. Wells were then incubated with biotinylated Siaα2-3Galβ1-4GlcNAc polyacrylamide array probes for 1 hour at room temperature, washed 3 times, incubated with streptavidin-alkaline phosphatase diluted 1:1000 in ELISA buffer, and washed again, and wells were developed with 100 μL substrate/well. (C) Neutrophil binding to immobilized α2-3–linked sialic acids requires Siglec-9. Neutrophils were incubated with BSAb, NBSAb, or PBS before carrying out the binding assay, as described in panel A. (D) Neutrophils bind to the α2-3–linked Sias of the GBS capsular polysaccharide via Siglec-9. Cell wall extracts were isolated from GBS strain COH1 using mutanolysin (Sigma-Aldrich, St Louis, MO) digestion as described.²³  Further purification of CPS from cell wall preparations was performed using ion exchange and size exclusion chromatography as described.²  The purified CPS was deposited on microtiter wells as in panel A. Neutrophils pretreated with the BSAb or NBSAb were added and allowed to adhere before washing away nonadherent cells. (E) Neutrophils bind to GBS cell surface extracts via Siglec-9. The unpurified type III GBS (COH1) cell surface extract, which includes the sialylated CPS, was immobilized in ELISA wells and studied for binding of neutrophils pretreated with the BSAb or NBSAb as in panel A. All results are representative of at least 3 experiments performed in triplicate. Neutrophil viability was approximately 85% at the end of the 30-minute time period of the assays, and the antibodies had no major effects on viability or activation (data not shown). Error bars represent SEM.