Quantifying SARS-CoV-2 Antigen Responses

Mouse splenocytes and lung cells were stimulated with antigens and assayed for production of IFN-γ as we published previously (6 (link)). Briefly, a single cell suspension of 1.0 × 10⁵ splenocytes or lung cells per well was seeded in U-bottom 96-well plates and incubated with T cell medium (Complete Advanced RPMI 1640 [Invitrogen] supplemented with 1 mM HEPES [Cellgro, Mediatech], 2 mM l-alanyl-l-glutamine [GlutaMAX Supplement, Gibco], 50 μM 2-mercaptoethanol [Sigma-Aldrich], 100 U/mL penicillin/100 μg/mL streptomycin, and 2% fetal bovine serum) alone or T cell medium supplemented with 2 μg/mL of N protein, 2 μg/mL of N peptide pool, or 2 μg/mL of M protein of SARS-CoV-2 or 5 × 10⁶ HI-LVS for 3 or 6 days. Afterwards, the culture supernatant fluid was collected, cell debris removed by centrifugation, and the supernatant fluid diluted 5-fold and stored in assay diluent (BD Biosciences) at −80°C until use. The production of mouse IFN-γ in the culture supernatant fluid was assayed using a mouse cytokine EIA kit (BD Biosciences) per the manufacturer’s instructions.