SARS-CoV-2 Plaque Assay in VeroE6-ACE2-TMPRSS2 Cells

Cells were grown in DMEM containing 10% (vol/vol) fetal calf serum (FCS) and incubated at 37°C in 5% CO₂. Plaque assays utilized VeroE6 expressing angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enhance virus entry [13 (link)]. Serial dilutions of sample were applied to cells for 1 hour at 37°C with rocking. Cells were overlaid with DMEM containing 2% FCS, 1.2% Avicel (DuPont, USA), 50 μg/mL Gentamycin (Fisher Scientific, UK), and 2.5 μg/mL Amphotericin-B (Sigma Aldrich, UK). After 72 hours, the overlay was removed, the monolayer washed, fixed with 100% methanol, stained with 25% (vol/vol) methanol and 0.5% (wt/vol) Crystal Violet, then washed, and plaques enumerated.