| Assay Method Information | |
| | SARS-CoV-2 Hela ACE2 EC50 determination and MERS-CoV Vero TMPRSS2 EC50 determination |
| Description: | Four thousand HeLa-ACE2 or Vero-TMPRSS2 cells (BPS Bioscience) were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 hours at 37°C, 5% CO2. Two hours before infection, the medium was replaced with 100 pL of DMEM (2% FBS) containing the compound of interest at concentrations 50% greater than those indicated, including a DMSO control. Plates were then transferred into the BSL3 facility and an MOI of 0.25 of SARS-CoV-2 or MERS-CoV was added in 50 pL of DMEM (2% FBS), bringingthe final compound concentration to those indicated. Plates were then incubated for 24 hours at 37 °C. After infection, supernatants were removed and cells were fixed with 4% formaldehyde for 24 hours prior to being removed from the BSL3 facility. The cells were then immunostained for the viral N protein (an inhouse mAb 1C7, provided by Dr. Andrew Duty, (Icahn School of Medicine at Mount Sinai), with a DAPI counterstain. Infected cells (488 nm) and total cells (DAPI) were quantified using the Cytation 1 (Biotek) imaging cytometer. Infectivity was measured by the accumulation of viral N protein (fluorescence accumulation). Percent infection was quantified as ((Infected cells/Total cells) - Background) *100 and the DMSO control was then set to 100% infection for analysis. Data was fit using nonlinear regression and IC50s for each experiment were determined using GraphPad Prism version 10.0.0 (San Diego, CA). Cytotoxicity was also performed using the MTT assay (Roche), accordingto the manufacturer’s instructions. Cytotoxicity was performed in uninfected cells with same compound dilutions and concurrent with viral replication assay. All assays were performed in biologically independent triplicates. |
| Affinity data for this assay | |
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