Antiviral Drug IC50 Determination

The IC₅₀ values for the tested compound were determined as previously described^{8 (link)}, with minor modifications. Briefly, the VERO-E6 monolayers in 96-well tissue culture plates were then washed once with 1 × PBS. The NRC-03-nhCoV” virus “TCID₅₀ = 100) was co-incubated with serial diluted working concentrations of the tested drugs at 37 °C for 1 h. The Vero-E6 cells were treated with virus/drug mixtures and kept at 37 °C for 1 h. Untreated/infected cells represented the virus control, however untreated/uninfected cells referred to the cell control. After 72 h of co-incubation at 37 °C in 5% CO₂ incubator, the cell monolayers were fixed with 100 μL of 10% formaldehyde for 20 min and stained with 0.1% crystal violet “in distilled water” for 15 min at RT. To dissolve crystal violet dye, 100 μL of the absolute methanol were added per well and the optical density of the color is eventually measured at 570 nm using the Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC₅₀ values were calculated using nonlinear regression analysis by plotting log inhibitor versus normalized response.