Lipofectamine 2000 transfectant reagent

To quantitate the cell surface expression of G7-HN (wt HN) and its mutants, BHK-21F cells were seeded into six-well plates one day prior to experimentation. Each well was transfected with 2 μg of wt HN or HN mutants by using the Lipofectamine 2000 Transfectant Reagent (Invitrogen, CA, USA). At 16 h post-transfection, each well was incubated with G7 polyclonal serum at a dilution of 1:200 for 1 h at room temperature. The monolayers were then washed extensively with PBS to remove unbound antibodies and incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-chicken IgG at a 1:500 dilution (Sigma-Aldrich, St. Louis, USA). Cells were washed extensively and resuspended in PBS containing 2% paraformaldehyde. The mean fluorescence intensity of 10,000 cells was recorded for each sample by using a FACSAria III flow cytometer (Becton Dickinson, New Jersey, USA)11 (link).

To observe the expression of HN protein, 1 μg of original HNs or mutant HNs was transfected into BHK-21 cells by using Lipofectamine 2000 Transfectant Reagent (Invitrogen, CA, USA). At 24 h post-transfection, each well was fixed with PBS containing 4% paraformaldehyde and blocked with PBS containing 1% bovine serum albumin (BSA). Then, the cells were incubated with an HN monoclonal antibody made by our laboratory at an optimized dilution of 1:1000 and incubated with Alexa Fluor^® 594-conjugated goat anti-mouse IgG (H + L) at a 1:500 dilution (Abcam, Shanghai, China) as the secondary antibody. Photographs of the cells were recorded under a fluorescence microscope (IX73; OLYMPUS, Tokyo, Japan). Empty cells were used as mock controls, and empty pCAGGS was used as the negative control. For rescued viruses, BHK-21 cells were incubated with viruses at an MOI of 0.1 for 12 h or 24 h. An IIFA at the virus level was performed with the methods described above.