Immunospot elispot plate scanner

Focus forming assay (FFA) were performed as described previously⁶¹ . Briefly, serial dilutions of tissue homogenate from inoculated mice were added to a monolayer of Vero-E6 cells in a 96-well plate. One hour after infection, cells were overlaid with 1% (wt/vol) methylcellulose in 2% fetal bovine serum (FBS), 1× minimal essential medium (MEM). 24 h after infection, plates were fixed for 15 min with 4% paraformaldehyde (PFA) followed by 1 hour with 10% neutral buffer formalin (NBF). Staining involved primary antibody polyclonal anti-SARS-CoV-2 guinea pig (BEI Resources – NR10361 1:15000) and secondary antibody goat anti-guinea pig–HRP (Thermo Cat# A16104 1:5000) in PermWash buffer (0.1% saponin, 0.1% BSA, in PBS). Treatment with TrueBlue peroxidase substrate (SeraCare – 5510–0030) produced focus-forming units that were quantified on an ImmunoSpot ELISpot plate scanner (Cellular Technology Limited).

Quantification of SARS-CoV-2 by FFA was performed by serial dilution of viral stocks or lung homogenate. Dilutions were added onto a monolayer of Vero cells in a 96-well plate. One hour after infection, cells were overlaid with 1% (w/v) methylcellulose in 2% FBS and 1× MEM. Plates were fixed for 30 minutes with 4% paraformaldehyde (PFA) 24 hours after infection. Staining involved 1° anti–SARS guinea pig serum (1:15,000; NR-10361, BEI Resources) and 2° goat anti–guinea pig HRP (200 ng/mL) in Perm Wash Buffer (0.1% saponin and 0.1% BSA in PBS). Treatment with TrueBlue peroxidase substrate (KPL, LGC Clinical Diagnostics) produced focus-forming units (FFU) that were quantified with an ImmunoSpot ELISpot plate scanner (Cellular Technology). For FRNT assays, serial dilutions of heat-inactivated serum from vaccinated mice were incubated with 100 FFU live SARS-CoV-2 (isolate USA-WA1/2020) for 1 hour at 37°C before infecting a monolayer of Vero cells in a 96-well plate. Viral foci were determined as above for FFAs.