| Assay Method Information | |
| | Measuring Antiviral Effect of Compounds |
| Description: | Vero E6 cells were selected for expression of the SARS CoV receptor (ACE2; angiotensin-converting enzyme 2) was used for the CPE assay. Cells were grown in MEM/10% HI FBS supplemented and harvested in MEM/1% PSG/supplemented with 2% HI FBS. Cells were batch inoculated with appropriate coronavirus (Toronto 2 SARS CoV-1 or USA_WA1/2020 SARS CoV-2) at M.O.I. 0.002, which results in 5% cell viability 72 (for SARS) hours post infection. Assay Ready Plates (ARPs; Corning 3712BC) pre-drugged with test compounds (30-90 nL sample in 100% DMSO per well dispensed using a Labcyte ECHO 550) were prepared in the BSL-2 lab by adding 5 μL assay media to each well. The plates were passed into the BSL-3 facility where a 25 μL aliquot of virus inoculated cells (4000 Vero E6 cells/well) was added to each well in columns 3-22. The wells in columns 23-24 contained virus infected cells only (no compound treatment). Prior to virus infection, a 25 μL aliquot of cells was added to columns 1-2 of each plate for the cell only (no virus) controls. After incubating plates at 37 C./5% CO2 and 90% humidity for 72 hours, 30 μL of Cell Titer-Glo (Promega) was added to each well. Luminescence was read using a Perkin Elmer Envision or BMG CLARIOstar plate reader following incubation at room temperature for 10 minutes to measure cell viability. Raw data from each test well was normalized to the average signal of non-infected cells (Avg Cells; 100% inhibition) and virus infected cells only (Avg Virus; 0% inhibition) to calculate % inhibition of CPE using the following formula: % inhibition=100*(Test Cmpd−Avg Virus)/(Avg Cells−Avg Virus). The SARS CPE assay was conducted in BSL-3 containment with plates being sealed with a clear cover and surface decontaminated prior to luminescence reading. |
| Affinity data for this assay | |
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