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Assay Method Information

Assay Name:  Viral titer plaque assay
Description:  Vero-E6 cells were seeded at 4 x 10E5 cells/well in 12-well plates and infected for 1 hour with the SARS-CoV-2 isolate USA-WA1/2020 at an MOI of 0.01. The cells were washed twice to remove residual unattached virus. Serial dilutions of each compound (0.1% DMSO in 2% FBS in DMEM media) were added to the cells and incubated at 37 C (2 dpi). After 2 dpi, the supernatant containing virus was cleared from cell debris at 1000 rpm for 10 min and frozen until analysis via plaque assay. For the plaque assay, Vero-E6 cells were seeded at 7.5 x 10E5 cells/well in 6-well plates. The following day, the media was removed and replaced with 100 μL of 10-fold serial dilutions of previously frozen viral supernatant. Plates were incubated at 37C for 1 hour with gentle rocking. Subsequently, overlay media (DMEM, 2% FBS, 0.6% Avicel RC-581) was added to each well. At 2 dpi for SARS-CoV-2 plates were fixed with 10% formaldehyde for 30 min, stained with crystal violet solution (0.5% crystal violet in 20% ethanol) for 30 min, and then rinsed with deionized water to visualize plaques.
Affinity data for this assay
 

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