2.2 Cell viability and cytotoxicity assays
For the cell viability assay, MDCK cells were cultured on a 96-well plate (2 × 104 cells/well) in Growth Medium. After incubation for 16 h at 37 °C, the cells were infected with each influenza virus at 103 50% tissue culture infectious dose (TCID50)/well and were washed with phosphate-buffered saline (PBS) at 1 h post-infection. Various concentrations (10 to 200 nM, n = 3 per dose) of aprotinin diluted with Infection Medium were added into each well to a final volume of 100 μl/well. After incubation for 72 h at 37 °C, cell viability was determined using the EZ-Cytox kit (Daeillab service Co., South Korea) according to the manufacturer’s instructions. Cell viability was indicated by percentage values, compared to the negative control (cells that were infected but not treated with aprotinin). Cytotoxicity of aprotinin was measured similarly as described above, but without infecting with the virus. Cytotoxicity was presented as a percentage value, compared to the negative control (wells containing cells only).