| Assay Method Information | |
| | SARS-CoV-2 antiviral assay |
| Description: | Antiviral activity of compounds against SARS-CoV-2 is evaluated as described in Xue, Xi et al.2020. Briefly, the human alveolar epithelial cell line (A549) is maintained in a high-glucose DMEM supplemented with 10% fetal bovine serum, 1% P/S and 1% HEPES (ThermoFisher Scientific). The A549-hACE2 cells that stably express human angiotensin-converting enzyme 2 (hACE2) are grown in the culture medium supplemented with 10 μg/mL Blasticidin S (Mossel E. C., et al 2005). Cells are grown at 37 °C with 5% CO2. All culture medium and antibiotics are purchased from ThermoFisher Scientific (Waltham, MA). All cell lines are tested negative for mycoplasma. A549-hACE2 cells (12,000 cells per well in phenol-red free medium containing 2% FBS) are plated into a white opaque 96-well plate (Corning). On the next day, 2-fold serial dilutions of compounds are prepared in DMSO. The compounds are further diluted 100-fold in the phenol-red free culture medium containing 2% FBS. Cell culture fluids are removed and incubated with 200 nL of diluted compound solutions and 50 μL of SARS-CoV2-Nluc viruses (MOI 0.025). At 48 h post-infection, 50 μL Nano luciferase substrates (Promega) are added to each well. Luciferase signals are measured using a Synergy Neo2 microplate reader. The relative luciferase signals are calculated by normalizing the luciferase signals of the compound-treated groups to that of the DMSO-treated groups (set as 100%). The relative luciferase signal (Y axis) versus the log10 values of compound concentration (X axis) is plotted in software Prism 8. The EC50 (compound concentration for reducing 50% of luciferase signal) are calculated using a nonlinear regression model (four parameters). Two experiments are performed with technical duplicates. |
| Affinity data for this assay | |
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