Neuraminidase from vibrio cholera

The presence of α2,3/α2,6-linked sialic acids on HEK-293T cells, chAEC, and dAEC was assessed by flow cytometry. Confluent monolayers were treated for 2 h at 37 °C in 5% CO₂ with fresh EGM^TM-2MV with or without 100 mU/mL neuraminidase from Vibrio cholera (Sigma-Aldrich, St. Louis, MO, USA), which enzymatically removes sialic acid moieties to act as negative control. Cells were washed, trypsinized, and incubated with PBS-2%FCS containing 10 µg/mL biotinylated Maackia amurensis lectin II (MAL-II; Vectorlabs, Burlingame, CA, USA; α2,3-sialic acid specific) or 10 µg/mL biotinylated Sambucus nigra agglutinin lectin (SNA; EY labs, San Mateo, CA, USA; α2,6-sialic acid specific) for 30 min at 4 °C. Biotin-labeled cells were stained with 5 μg/mL FITC-conjugated streptavidin (F0422; Agilent, Santa Clara, CA, USA) for 30 min at 4 °C, detected by flow cytometry, and analyzed using FlowJo v10.7.2 software (BD Biosciences, Ashland, OR, USA).

Reticulocyte-enriched samples were prepared with up to 50% hematocrit. The RBCs were washed with 500 μl of incomplete RPMI 1640 medium twice by centrifugation at 500× g for 3 min at 4 °C. The RBCs were incubated with neuraminidase (from Vibrio cholera, Sigma-Aldrich), trypsin (from bovine pancreas, Sigma-Aldrich) and chymotrypsin (from bovine pancreas, Sigma-Aldrich) at 37 °C on a rotator for 1 h. The trypsin and chymotrypsin-treated RBCs were incubated with a trypsin inhibitor (from the Glycine max soybean, Sigma) at 37 °C for 10 min. The RBC samples were washed twice with 10 ml of incomplete RPMI 1640 medium. The packed cells were prepared at a concentration of 1 × 10⁶cell/ml and used for flow cytometry analysis.