Virus Quantitation via Focus Forming Assay

Focus forming assays (FFAs) were performed as previously described [⁵⁰]. Briefly, Vero E6 cells were seeded in 96-well plates to be 100% confluent. Samples were serially diluted in serum-free media and 20 μl was used to infect cells. Cells were incubated for 45 min at 37°C with 5% CO₂ before 0.85% methylcellulose overlay was added. Cells were incubated for 24 h at 37°C with 5% CO₂. After incubation, overlay was removed, and cells were washed three times with PBS before fixed and virus inactivated by 10% formalin for 30 min at room temperature. Cells were then permeabilized and blocked with 0.1% saponin/0.1% BSA in PBS before incubated with α-SARS-CoV-2 Nucleocapsid primary antibody (Cell Signaling Technology) at 1:1000 in permeabilization/blocking buffer overnight at 4°C. Cells were then washed (3x) with PBS before incubated with Alexa Fluor^™ 555-conjugated α-mouse secondary antibody (Invitrogen #{"type":"entrez-protein","attrs":{"text":"A28180","term_id":"80983","term_text":"pir||A28180"}}A28180) at 1:2000 in permeabilization/blocking buffer for 1 h at room temperature. Cells were washed (3X) with PBS. Fluorescent foci images were captured using a Cytation 7 cell imaging multi-mode reader (BioTek), and foci were counted manually.