Quantifying SARS-CoV-2 Spike Protein Expression

293T cells were co-transfected with a GFP expressor (pIRES2-GFP, Clontech, Mountain View, CA, USA) in combination with plasmids encoding the full-length S of SARS-CoV-2 variants; 48 h post transfection, S-expressing cells were stained with the CV3-25 Ab [28 (link)] or plasma (1/500 dilution). AlexaFluor-647-conjugated goat anti-human IgG Abs (1/1000 dilution) was used as a secondary Abs. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on an LSRII cytometer (BD Biosciences, San Jose, CA, USA) and data analysis was performed using FlowJo v10.7.1 (Tree Star). The seropositivity threshold was established using the following formula: (mean of pre-pandemic SARS-CoV-2 negative plasma + (3 standard deviation of the mean of pre-pandemic SARS-CoV-2 negative plasma). The conformational-independent S2-targeting mAb CV3-25 was used to normalize S expression. CV3-25 was shown to effectively recognize all SARS-CoV-2 S variants [34 (link)].