High-Content Screening of SARS Inhibitors

Vero cells (1.2 × 10⁴ cells) were cultured in DMEM with 1× antibiotic–antimycotic solution and 2% FBS in 384-well black culture plates. The serially diluted compounds and 0.0125 multiplicity of infection (MOI) SARS-CoV-2, 0.05 MOI SARS-CoV, or 0.0625 MOI MERS-CoV were added. At 24 h post-infection, the cells were fixed with 4% paraformaldehyde and stained with antibodies against the SARS-CoV-2 nucleocapsid protein, SARS-CoV spike protein, or MERS-CoV spike protein (Sino Biological Inc., Beijing, China), followed by goat antirabbit IgG secondary antibody and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). Images were analyzed using the Operetta^® High-Content Imaging System (20×; PerkinElmer, Inc., Waltham, MA, USA) and Image-Mining 3.0 plug-in software [20 (link)].