2.7. Analysis of GFP Expression Using Flow Cytometry

MDCK or A549 cells were cultured in 24-well plates (1 × 10⁵ cells/well) for 18 h. A/PR/8/34 (MOI = 1) was mixed with different concentrations of RVS and RVSE (0, 12.5, 25, 50, and 200 μg/mL), and the mixtures were incubated at 37°C for 1 h. MDCK and A549 cells were infected with these mixtures at 37°C for 2 h. Subsequently, the virus was removed, and the cells were washed three times with PBS, and the medium was replaced by complete DMEM. Cells were incubated for 24 h at 37°C with 5% CO₂. Reduction of viral infection was determined by measuring GFP expression using flow cytometry. MDCK or A549 cells were harvested and resuspended in 1 mL of PBS containing 2% FBS and fixed in suspension with 4% paraformaldehyde. The cells were washed three times with PBS and stored at 4°C until analysis with a CytoFLEX flow cell counter (Beckman). We analyzed data using FlowJo software.