2.7 Dose-response curve (DRC) analysis by immunofluorescence assay
Vero cells were seeded at 1.2 × 104 cells per well in black, 384-well, μClear plates (Greiner Bio-One, Austria). After 24 h, the cells were transferred into the BSL-3 containment facility and SARS-CoV-2 was added at a multiplicity of infection (MOI) of 0.008 and incubated for additional 24 h. The cells were then fixed with 4% PFA and immunostained with an antibody against SARS-CoV-2 nucleocapsid (N) protein, and cell nuclei were visualized with DNA fluorochrome Hoechst 33342. The images were acquired using Operetta high-throughput imaging device (Perkin Elmer) and analyzed using Columbus software (PerkinElmer, Inc. Waltham, MA) to quantify cell numbers and infection ratios. Antiviral activity was normalized to infection control (0.5% DMSO) in each assay plate. DRCs were generated using Prism software (GraphPad). IC50 values were measured in duplicates and calculated using nonlinear regression analysis.