Quantifying NA protein expression on MDCK-II cells

The expression of NA protein on MDCK-II cells was quantified by flow cytometry using FACS analysis as previously described with few modifications [65 (link)]. Briefly, MDCK-II cells in 24-well plates were infected with MOI 1 of different viruses in three replicates. At 8 and 24 h, the supernatant (0.5mL) were firstly collected then cell sheets were dissociated by trypsin (0.250 mL) and anti-N1 polyclonal antibodies were added at a ratio 1:1000 for 1 h to ~5x10⁶ non-permeabilized single cells. After washing, secondary anti-rabbit antibodies were added 1:20000 and the cells were subjected to immuno-staining for flow cytometry analysis using a BD FACSCanto flow cytometer (BD Biosciences). The mean fluorescence intensity (MFI) of non-infected cells was subtracted from infected cells and the results were shown as ΔMFI and standard deviation of all replicates. For the detection of SA moieties, MDCK-II and MDCK-SIAT cells were infected at an MOI of 1 with rg-AL or rg-HL-16. After 2 hour incubation at 37°C cells were detached with trypsin and washed once with staining buffer. Fluorescein-labelled Sambucus nigra lectin (SNA; Vector Laboratories) and biotin-labelled Maackia amurensis lectin II (MAL II; Vector Laboratories) were used for the detection of (α-2,6) or (α-2,3) linked sialic acids, respectively, in infected and non-infected cells. After washing, MAL II binding was detected using PE-Cy7-labelled streptavidin (Biolegend). All incubation steps were carried out at 4°C in the dark. Stained infected and non-infected cells were treated with fixation buffer according to the manufacturer’s protocol (Biolegend). Reading was done using a BD FACSCanto flow cytometer (BD Biosciences). The results are the average and standard deviation of a triplicate experiment.