Flow cytometry - Measurement of SARS-CoV-2 reactive T cells
As previously described, PBMCs were plated in 96-U-Well plates in RPMI 1640 media (Life Technologies) (22,23). Each well was stimulated with wildtype (WT) S-protein (Miltenyi Biotec) or left untreated as a control for 16 h. The proteins were dissolved per manufacturer’s directions. As a positive control, cells were stimulated with staphylococcal enterotoxin B (1 μg/mL, Sigma-Aldrich). After 2 h, brefeldin A (1 μg/mL, Sigma-Aldrich) was added. Detailed listing of the antibody panels for general phenotyping and T cell activation ex vivo is in Table S2. The PBMCs stimulated overnight were stained with optimal concentrations of antibodies for 10 min at room temperature in the dark. Stained cells were washed twice with PBS/BSA before preparation for intracellular staining using the Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific) as per the manufacturer’s instructions. Fixed and permeabilized cells were stained for 30 min at room temperature in the dark with an optimal dilution of antibodies against the intracellular antigen. All samples were immediately acquired on a CytoFLEX flow cytometer (Beckman Coulter). Quality control was performed daily using the recommended CytoFLEX daily QC fluorospheres (Beckman Coulter). No modification to the compensation matrices was required throughout the study. Antigen-reactive responses were considered positive after the non-reactive background was subtracted, and more than 0.01% were detectable. Negative values were set to zero.