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.