2.4. Plaque Assay

Plaque assays were performed in Vero E6 cells (ATCC CRL-1586), using a modified protocol originally developed by De Madrid and Porterfield [20]. In Vero E6 cells, SARS-CoV-2 forms large, circular plaques, which are regular in shape and size, and, therefore, this cell line is particularly suitable for plaque assays and subsequent viral titer quantification. Briefly, 10-fold dilutions of virus were prepared in 24-well tissue culture plates, and the cells were added to each well (0.6–1.5 × 10⁵ cells/well). After 4 h incubation, the suspension was overlaid with 1.5% (w/v) carboxymethylcellulose in DMEM. Following a 5-day incubation at 37 °C, the infected plates were washed with phosphate-buffered saline, and the cell monolayers were stained with naphthalene black. The virus titer was expressed as plaque forming units (PFU)/mL.