2.8. Plaque Reduction Assay

Plaque reduction assays were performed as described previously [52]. Briefly, confluent monolayers of Vero E6 ACE2 cells were cultured in a 6-well tissue culture plate and infected with approximately 200 pfu of SARS-CoV-2 WA1/2020 strain. After 45 min on a rocker, the viruses were removed and replaced by overlay medium (DMEM containing 1% low-melting agarose and test compounds at different concentrations). After incubating the cultures for 3 d, the monolayers were fixed with 4% (v/v) formaldehyde solution and the agarose plugs were removed and stained with 1% (w/v) crystal violet solution. The concentration required to deduce the EC₅₀ was calculated by regression analysis of the dose–response curves generated from these data [53]. The experiments were performed three times.