SARS-CoV-2 Plaque Assay Protocol

As previously described [35 (link)], Vero cell monolayers in six-well plates were inoculated with 250 pfu per well of virus and incubated at 32°C for seven days with an overlay containing 0.8% methylcellulose. On day 7, plates were fixed with cold 80% methanol and incubated with a cocktail of three anti-RSV F MAbs [28 (link)] in Odyssey Blocking Buffer in PBS (Li-Cor), washed, and incubated with an R-phycoerythrin goat anti-mouse IgG(H+L) secondary antibody (Thermofisher, P-852). Plaques were visualized using the Celigo imager (Nexcelcom Bioscience) and the plaque size (μm²) was analyzed using Celigo software. An average of 3451 (±1200) individual plaques were analyzed per virus.