Fig 1.

Detection and quantification of α-2,3- and α-2,6-linked sialic acid (SA) receptors on the cell surface of pulmonary microvascular endothelial cells. (A) Fluorescence lectin staining. Primary human lung blood microvascular endothelial cells (HMVEC-LBI) and human lung microvascular endothelial cells (HULEC) were grown on collagen-coated chamber slides overnight. The cells were incubated with no lectin (control), biotinylated SNA, MAA I, or MAA II, followed by FITC-conjugated avidin D (green) and DAPI (blue) for nuclei. (B) Trypsinized endothelial cells were incubated with FITC-conjugated SNA or MAA (5 μg/ml) and analyzed using flow cytometry. Fluorescent intensities for the α-2,6-SA configuration (SNA, green) and the α-2,3-SA configuration (MAA, blue) were compared to that for unstained cells (red). (C) Virus binding assay. HMVEC-LBI cells grown on collagen-coated chamber slides were incubated with FITC-labeled influenza virus at comparable HAU titers, followed by anti-FITC–horseradish peroxidase (HRP) antibody and AEC detection. The images were taken using the same exposure. (D) Fluorescent lectin staining. Primary rat pulmonary microvascular endothelial cells (PMVEC) and primary rat pulmonary arterial endothelial cells (PAEC) were stained green.