Immunofluorescence (IF) staining of influenza A virus-infected MDCK cells

The MDCK cells were seeded on a 96-well plate at the density of 1 × 10⁴ cells/well. The Y30 extract (final 3.1–12.5%) was mixed with A/PR/8/34 or A/Aichi/2/68 viruses at a MOI of 0.1 in the infection medium and incubated for 30 min at 37°C in the presence of 5% CO₂. Each mixture was added to the cells at 37°C in the presence of 5% CO₂. The cells were treated with the Y30 extract (final 3.1–12.5%) for 24 h at 37°C in the presence of 5% CO₂. Subsequently, the Y30-treated cells were washed prior to the addition of the A/PR/8/34 virus at a MOI of 0.1 in the infection medium. Water (final 3.1–12.5%) or bakuchiol (final 3.1–12.5 μM) were used as the negative or positive controls, respectively. After influenza A virus infection for 24 h, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 4°C and subsequently permeabilized with 0.3% Triton X-100 for 20 min at 25°C. A mouse primary antibody was used to detect the NP of A/PR/8/34 or A/Aichi/2/68 viruses (FluA-NP 4F1; SouthernBiotech, AL, USA). An Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) antibody (Thermo Fisher Scientific) was used as the secondary antibody. Cell nuclei were then stained using diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Wells were photographed using a fluorescence microscope (BIOREVO BZ-X700, Keyence, Osaka, Japan). The percentage of influenza A NP-positive cells per DAPI-positive cells were calculated based on measurements recorded using the BZ-X Analyzer software (Keyence).