White 96 well plates

Viral titrations were performed largely as previously described^{51 (link)}. VeroE6-TMPRSS2 cells were seeded at a density of 60,000 cells/well into white 96-well plates (Falcon; #353296). 24hrs later, viral samples of interest were serially diluted 10-fold in DMEM + 10% FBS. Plates were aspirated and infected with diluted viral samples for 1hr at 37°C and 5% CO₂ before being overlaid with a 1.2% methylcellulose (Acros; #332620010) solution suspended in DMEM. After 24hrs, the methylcellulose solution was aspirated, and the plates were fixed a 4% formaldehyde solution (Honeywell; #F1635–4L) for 20mins before being washed once with deionized (DI) water. Cells were permeabilized in 50ul of 0.05% Triton X100 (Fisher; #BP151–100) for 5mins, washed with PBS and blocked (5% non-fat milk solution in PBS) for 1hr at room temperature. Virally infected cells were detected with an anti-SARS-CoV-2 N antibody (Sinobiological; #40143-R001, 1:20,000) resuspended in 5% milk for 1hr at 37°C. Cells were washed twice with PBS and stained with a HRP-conjugated secondary antibody (Seracare; #5220–0337, 1:4,000) resuspended in 5% milk for 1hr at 37°C. Cells were washed twice with PBS, and foci developed using a TruBlue HRP substrate (SeraCare; #5510–0030). Foci were imaged on a BioTek ImmunoSpot S6 MACRO Analyzer and manually counted.