SARS-CoV-2 viral quantification via antibody staining in presence of inhibitors
(Mount Sinai, New York). Two thousand (2,000) Vero E6 cells were seeded into 96-well plates and incubated for 24 hours. Two hours before infection, the medium was replaced with 100 μL 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 BSL-3 facility and 100 PFU of SARS-CoV-2 (MOI 0.025) was added in 50 μL of DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incubated for 48 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 BSL-3 facility. The cells were then immunostained for the viral NP protein (anti-sera produced in the Garcia-Sastre lab; 1:10,000) with a DAPI counterstain. Infected cells (488 nM) and total cells (DAPI) were quantified using the Celigo (Nexcelcom) imaging cytometer. Infectivity was measured by the accumulation of viral NP protein in the nucleus of the Vero E6 cells (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. The IC50 for each experiment was determined using the Prism software (GraphPad). For select inhibitors, infected supernatants were assayed for infectious viral titer using the Median Tissue Culture Infectious Dose TCID50 method. For this, infectious supernatants were collected at 48 hours post infection and frozen at −80°C until later use. Infectious titers were quantified by limiting dilution titration on Vero E6 cells. Briefly, Vero E6 cells were seeded in 96-well plates at 20,000 cells/well. The next day, SARS-CoV-2-containing supernatant was applied at serial 10-fold dilutions ranging from 10−1 to 10−6 and, after 5 days, viral CPE was detected by staining cell monolayers with crystal violet. TCID50/mL was calculated using the method of Reed and Muench.