Journal: Frontiers in Immunology

Article Title: Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs

doi: 10.3389/fimmu.2022.827760

Figure Lengend Snippet: Binding of biotinylated lectins and viruses to human PBMCs. The MDCK and CHO cells and PBMCs were incubated for 1 hour with (A) biotinylated Sambucus nigra lectin (SNA, final concentration 0.4 µg/ml) or biotinylated Maackia amurensis lectin 1 (MAL-1, final concentration 1 µg/ml) at room temperature or (B) with biotinylated IAVs Mem-H1N1(final concentration 200 µg/ml) or Mal-H1N1 (final concentration 10 µg/ml) at 4 °C. Lectin or virus binding was detected as mean fluorescence intensity (MFI) after staining with streptavidin–APC. Cells were then fixed and stained for subpopulation as described in Material and Methods and subjected to flow cytometric analysis. Data of one representative donor are shown.

Article Snippet: For staining with plant lectins, the cells were incubated for 1 h at room temperature with biotinylated Sambucus nigra lectin (SNA) (Vector Labs, B-1305, final concentration 0,4 µg/ml) or Maackia amurensis lectin (MAL I) (Vector Labs, M-1315, final concentration 1 µg/ml) and washed twice with the lectin buffer (0.01 mM MnCl 2 , 0.1 mM CaCl 2 , 0.1 mM MgCl 2 , 0.1 % BSA in PBSdef).

Techniques: Binding Assay, Incubation, Concentration Assay, Fluorescence, Staining

Journal: Nature Communications

Article Title: Human-type sialic acid receptors contribute to avian influenza A virus binding and entry by hetero-multivalent interactions

doi: 10.1038/s41467-022-31840-0

Figure Lengend Snippet: a Graphical summary of methods that were employed to establish density gradients on HEK ΔSia cells and LAMP I. The Sia repertoire exposed on HEK ΔSia cells was specifically tuned by transfection with different concentrations of ST6Gal1 and/or ST3Gal4. Co-transfected LAMP I will be secreted in a C-terminally biotinylated form that can be attached to streptavidin BLI sensors for comparing entry into HEK ΔSia cells with binding to LAMP I with a similar sialylation pattern in a BLI assay. b , c Comparison of virus binding rates to avian-type and human-type receptors. Absolute binding rates (nm/300 s), normalized to a particle number of 10 9 particles of each virus strain, were determined. b Binding to sensors fully loaded with LAMP I produced in HEK ΔSia cells co-transfected with 25 ng ST6Gal1 or 25 ng ST3Gal4 yielding high levels of LacNAc termini carrying 2-6Sia (magenta bars) or 2-3Sia (black bars). c Binding to sensors fully loaded with 2-6S(LN)2 (magenta bars) or 2-3S(LN)2 (black bars). Relative binding rates were plotted as a function of relative receptor density for human H3N2 strains BK79 ( d ) and WU95 ( e ), avian H5N1 strain HU02 ( f ) and human H1N1 strain PR8 ( g ). Black lines show binding to LAMP I proteins obtained by co-expression with a concentration range of sialyltransferases ST6Gal1 ( d , e ) or ST3Gal4 ( f , g ). Magenta lines show binding to LAMP I proteins obtained by co-expression of a concentration range of ST6Gal1 in combination with 25 ng ST3Gal4 ( d , e ) or a concentration range of ST3Gal4 in combination with 25 ng ST6Gal1 ( f , g ). 2-6Sia density or 2-3Sia density on LAMP I was quantified by SNA or MAL I binding respectively and plotted on the x-axis as relative SNA ( d , e ) or MAL I ( f , g ) signal reflecting the relative receptor density. Data from biological replicates were fitted into single curves of which R-sq values are indicated. ( b n = 7, 3, 8, 8, 3, 3, 4, 4, 3, 3, 9 and 4 for each bar from left to right; c n = 6, 6, 4, 8, 3, 3, 4, 7, 3, 3, 5 and 4 for each bar from left to right). Data are presented as mean values +/−S.D. Panel a was Created with BioRender.com. Source data are provided as a Source Data file.

Article Snippet: Cells firstly were incubated with biotinylated-lectins (MAL I, Vector Labs; SNA, Vector labs) for 1 hr and then complexed with Streptavidin for 1 hr, (Alexa Fluor ™ 568 conjugate (1 mg/mL),Thermo Fisher).

Techniques: Transfection, Binding Assay, Produced, Expressing, Concentration Assay