Ultramdck media

MDCK cells (100 µL/well) were seeded at a concentration of 2 × 10⁵ cells/mL in 96-well cell culture plates (Sigma) and incubated at 37 °C for 12 h. The mAbs were diluted to a starting concentration of 30 µg/mL in PBS and serially diluted 1:2 in UltraMDCK media (Lonza) supplemented with tosyl phenylalanyl chloromethyl ketone-treated trypsin (infection media; Sigma) at a concentration of 1 µg/mL, in 96-well cell culture plates (Sigma). The viruses were diluted to a concentration of 100 × TCID₅₀/50 µL (A/Hunan/02285/2017 (Hunan), A/feline/New York/16-040082/2016 (New York), A/Hong Kong/2014/2017 (Hong Kong)) in infection medium. Next, 60 µL of virus dilution was incubated with 60 µL of mAb serial dilution and incubated on the shaker at room temperature for 1 h. The plates were incubated at 33 °C for 48h  (New York) or 72 h (Hunan, Hong Kong). The readout was performed by the means of classical hemagglutination assay. This readout was chosen because it is more objective than assessment of cytopathic effects but easier to perform than staining for virus antigen (e.g., for nucleoprotein). In brief, chicken RBCs (Lampire Biological Laboratories) was diluted to a concentration of 0.5% in PBS and added to 50 µL of cell supernatant in v-bottom plates (Corning). After 45–60 min the plates were scanned and the results analyzed in Microsoft Excel and GraphPad Prism 7.

A549 cells were maintained in DMEM supplemented with 10% FBS, 10 U ml⁻¹ penicillin, and 10 mg ml⁻¹ streptomycin) and were plated in 96-well, white-walled plates (Costar) at 2.5 × 10⁵ cells per ml overnight at 37 °C with 5% CO₂. The following day, cells were washed with PBS and infected with A/Netherlands/602/2009 at a multiplicity of infection of 5 in UltraMDCK media (Lonza) for 24 h in the absence of TPCK-treated trypsin. mAbs were serially diluted in assay buffer (RPMI 1640 supplemented with 4% ultra-low IgG FBS; Gibco), starting at 60 μg ml⁻¹ and diluted threefold. Cell medium was aspirated and 25 μl of assay buffer and 25 μl of diluted antibody were added to each well. Jurkat cells expressing human FcgRIIIa with a NFAT-driven luciferase reporter gene (Promega) were diluted to 3 × 10⁶ cells per ml, 25 μl of cells was added to each well and incubated at 37 °C with 5% CO₂ for 6 h. Plates were removed from the incubator and placed at room temperature for 15 min. Of the BioGlo luciferase substrate (Promega), 75 μl was added to each well and luminescence was read immediately using a Syngery H1 hybrid multimode microplate reader (Biotek). EC₅₀ values were determined using Prism 8 (GraphPad).