Cells were seeded at a density of 30,000 cells per well in a flat-bottomed 96-well cell culture plate and incubated overnight at 37°C to adhere. Cells were washed twice with 200 μl of phosphate buffered saline (PBS), and 50 μl of neuraminidase (Sigma-Aldrich, cat. no. 11080725001) was added to the appropriate wells at a concentration of 50 mU/ml, diluted in serum-free media, and incubated for 1 hour at 37°C. Cells were cooled on ice and washed twice with 200 μl of PBS. GFP-expressing, replication-incompetent viruses were added to the appropriate wells at a concentration of 2000 or 5000 VP per cell, in 100 μl of serum-free media, at 4°C, and incubated on ice for 1 hour. Serum-free media alone were added to uninfected control wells. Cells were washed twice with 200 μl of cold PBS, and complete media were added (DMEM, 10% fetal calf serum) and incubated for 48 hours at 37°C. Cells were then trypsinized and transferred to a 96-well V-bottom plate, washed twice in 200 μl of PBS and fixed in 2% paraformaldehyde for 20 min before wash, and resuspended in 200 μl of PBS.

Samples were run in triplicate and analyzed by flow cytometry on Attune NxT (Thermo Fisher Scientific), analyzed using FlowJo v10 (FlowJo, LLC), gating sequentially on singlets, cell population, and GFP-positive cells. Levels of infection were described in terms of total fluorescence, defined as the percentage of GFP-positive cells (% + percentage positive) multiplied by the median fluorescence intensity of the GFP-positive population.

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