C9481

SARS-CoV-2 was titrated with virus plaque assay on Vero E6 cells. Virus stocks were serially diluted in virus titration media (VTM, DMEM supplemented with 5% fetal calf serum, 2 mM l-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin) and titrated on 24-well plates. The virus inoculum was added on the Vero E6 cells and incubated at 37°C. After 1 h, the inoculum was removed and the cell were overlaid with VTM supplemented with 1.5% carboxymethylcellulose (medium viscosity, C9481, Sigma-Aldrich) and incubated at 37°C. The overlay was removed from the cell at 3 hpi and the plates were fixed by submerging them in a tank of 6% formaldehyde (methanol stabilized) for at least 1 h. The cell monolayer was stained with crystal violet and the plaques were quantified by visual inspection with microscope. Virus supernatants were titrated in a similar fashion on 96-well plates.
For plaque purification, Vero E6 cells seeded in 24-well plate were infected with 10 PFU of early passage FI-P4 strain. After 1 h of infection, the inoculum was removed, and cells were overlaid with VTM supplemented with 1.5% carboxymethylcellulose and incubated at 37°C for 3 days. Plaques were identified under the inverted microscope and well-separated plaques were picked by scratching with 200-μL pipette tips. The tips were transferred to Vero E6 cells in a T25 flask to recover the virus.