Arthrobacter ureafaciens sialidase aus

Approximately 3 ml of venous blood (AA and SS) was collected in EDTA, centrifuged, and erythrocytes isolated. Approximately 2 million erythrocytes were incubated with 10μg of rhSiglec-9 protein (R&D Systems) for 30 minutes on ice. Erythrocytes were then stained with either anti-CD235a (Biolegend) or anti-IgG (Biolegend) on ice for 30 minutes and analyzed by flow cytometry. One set of AA erythrocytes were pretreated with 20mU of Arthrobacter ureafaciens sialidase (AUS) (Sigma-Aldrich) for 15 minutes at RT followed by protein incubation and staining [16 (link)].

Cell monolayers on glass coverslips were fixed with PFA (3.7% in PBS, 15 min), either left non-permeabilized or permeabilized with PBS, 0.1% Triton X-100 (10 min), successively incubated (1 hr each) with blocking buffer (PBS, 0.05% Tween-20, 2% BSA) and with HE⁰-Fc lectins (MHV-S, 50 µg/l; PToV-P4, 20 µg/ml; ECoV-NC99, 100 µg/ml; BCoV-LUN, 100 µg/ml; concentrations used were 2- to 4-fold above saturation as determined by LBA with 2-fold serial dilutions of each lectin), and then for 30 min with goat-α-human IgG-Dylight488 and/or goat-α-bovine IgG-Dylight549 (Jackson ImmunoResearch; 1:100), and with Hoechst-33258 (1:200). All incubations were in blocking buffer. Washing was performed with PBS, 0.05% Tween-20. The cells, embedded in FluorSave (Calbiochem), were examined by standard fluorescence microscopy (Leica DMRE).
Inhibitors were added to culture supernatants of HRT-18 cells (Benzyl-Gal-NAc, 2.5 mM; NB-DNJ, 0.2 mM; NB-DGB, 0.1 mM; NB-DMJ, 0.1 mM). Incubation was continued for 72 hr. Cells were fixed and stained with PToV-P4 and BCoV-LUN HE⁰-Fc lectins as described above. To de-O-acetylate or remove cell-surface Sias prior to L-IFA, PFA-fixed Ederm cells were incubated for 2 hr at 37°C with PBS, containing 20 µg/ml MHV-S HE⁺-Fc or 100 mU/ml Arthrobacter ureafaciens sialidase (AuS; Sigma-Aldrich), respectively.