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Virus titration by TCID50 calculation
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The day prior to infection, 50,000 VeroE6 cells per well were seeded in 96-well tissue culture plates using 10% FBS DMEM, and then incubated overnight at 37 ◦C in a humidified, 5% CO2 atmosphere-enriched chamber. On the day of infection, serial 2.5-fold dilutions (from 10−1 to 10−6.5) of the A549 cell culture supernatant were prepared in DMEM and used to infect Vero E6 cells; each dilution was tested in four replicates. The plates were incubated for at least 96 h and observed to monitor the development of cytopathic effect (CPE) using an EVOS Floid microscope (Invitrogen). Viral titers, expressed as TCID50/mL, were calculated according to both Reed and Muench and Karber methods based on three or four replicates for dilution (Reed and Muench, 1938).
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