Tf perm wash buffer

Collected cells were fixed in 70% (v/v) ethanol overnight at 4°C. Cells were subsequently washed three times with 1× TF Perm/Wash Buffer (BD Biosciences, 51-9008102) and then stained in 1× TF Perm/Wash Buffer with 50 μg/ml propidium iodide (Sigma, P4864) and 0.1 mg/ml RNase A (Sigma-Aldrich, 10109142001). Cell cycle data was acquired using LSR II cytometer (BD Biosciences) and analyzed with FlowJo (v.10.1).

Cultured T cells were stimulated separately with the SARS-CoV-2 overlapping peptide pools, corresponding to ORF3a, N, M and S proteins from SARS-CoV-2, and incubated at 37°C for 4 hours in the presence of GolgiPlug. Following stimulation, cells were washed and stained with anti-CD8-BV786, anti-CD4-PE-Cy7, anti-CD27-PE/Dazzle 594, anti-CD28-BV480 (BD Biosciences), anti-CD57-BV605, anti-CD45RA-FITC (BD Biosciences) and live/dead fixable near-IR dead cell stain (Life Technologies) for 30 minutes at 4°C before being fixed and permeabilized with TF Fix/Perm buffer (BD Pharmingen). After 1 hour of fixation, cells were washed in TF Perm/Wash buffer (BD Pharmingen) and stained with anti-TNF-APC (BD Biosciences), anti-Tbet-PE (eBioscience), anti-Eomes-PerCP-eFluor 710 (eBioscience), anti-Granzyme B-AF700 (BD Biosciences) and anti-Perforin-BV421 (Biolegend) for a further 30 minutes. Finally, cells were washed again and acquired using a BD LSRFortessa with FACSDiva software. Post-acquisition analysis was performed using FlowJo software (TreeStar). Gating analysis for the detection of antigen-specific TNF-producing cells is outlined in S1 Fig. TNF-producing cells from positive assays were then concatenated and t-SNE analysis was performed using the following parameters (Eomes, T-bet, CD27, CD28, CD57, granzyme B and perforin).