Plaque forming unit assay (PFU)

PFU assays were used to titer viral stocks and to determinate titer of Mito-C antiviral effect in infected cells. Briefly, supernatants from these two experiments were serial diluted following 1:3 dilution factor in infection medium. 500μL of each virus dilution was added to a monolayer of MDCK cells in 6-well plates. Cells were incubated 90 min for viral uptake and then virus was washed off. A mix of 2% SeaKem agarose (Lonza, #50011) and 2xMEM (Gibco) supplemented with 0.4μg/ml TPCK trypsin (1:1) were added to each well. Plates were incubated for 72 h at 37°C, 5%CO₂. Cells were fixed in a 10% PFA solution and then stained with crystal violet solution (0.3% crystal violet, 10% ethanol in water) for 30min at room temperature and abundantly rinsed with distilled water. Infectious titers were calculated considering the corresponding dilution factor after PFU numeration.