Quantification of SARS-CoV-2 Variant Infection

The supernatants were collected from infected cells at indicated time points. The supernatants were ten-fold diluted using growth medium with 2% FBS. After 1h of viral adsorption, virus inoculum was removed. Overlay medium with 1.5% carboxymethylcellulose (CMC) for all SARS-CoV-2 variants except for Omicron where 1% CMC was used. The plates were incubated at 37° C for 28 h for omicron and 24 h for all other SARS-CoV-2 variants. After incubation time, cells were fixed with formaldehyde solution followed by permeabilization with IMF buffer for 20 min incubation. Further, cells were stained with anti-spike RBD rabbit polyclonal antibody dilution at a dilution of 1:2000 for 1 h, followed by incubation with secondary antibody i.e., Alexa flour 488-conjugated anti-rabbit antibody at 1:500 dilution for 1 h. For omicron isolate, anti-nucleocapsid primary antibody was used at a dilution of 1:2000. This was followed by incubation with secondary antibody i.e., Alexa flour 488-conjugated goat anti-mouse IgG secondary antibody at 1:500 dilution. Fluorescent foci indicating infected cells were observed and counted using AIDiSpot reader using FITC channel.