Facsdiva v9

Blood was collected in EDTA tubes and 100 µL of whole blood was aliquoted for flow cytometry analysis. Whole blood was lysed with 1 mL of red cell lysis buffer for 10 min at room temperature. Cells were washed with 1 mL PBS and centrifuged at 350 × g for 5 m. Following two more washes, cells were resuspended in PBS for viability staining using near infra-red viability dye according to the manufacturer’s instructions. The viability dye reaction was stopped by the addition of FACS buffer (2% heat-inactivated FCS in 2 mM EDTA) and cells were centrifuged at 350 × g for 5 min. Cells were then resuspended in human FC-block according to manufacturer’s instructions for 5 min at room temperature. The whole blood cocktail (Supplementary Table 3) made up at 2X concentration were added 1:1 with the cells and incubated for 30 min on ice. Following staining, cells were washed with 2 mL FACS buffer and centrifuged at 350 × g for 5 m. Cells were then resuspended in 2% PFA for a 20 min fixation on ice, washed, and resuspended in 150 µL FACS buffer for acquisition using the BD LSR X-20 Fortessa and BD FACS DIVA V 9.0 software. For all flow cytometry experiments, compensation was done at the time of sample acquisition using compensation beads. Supplementary Figure 3 depicts the manual gating strategy for whole blood samples.

Labeled cells were counted by flow cytometry using a BD FACSVerse analyzer. For flow cytometric analyses, labeled cells were counted and analyzed using BD FACSuite v1.0.6 and BD FACS Diva v9.0 software. Cells expressing GFP were excited at 488 nm, and red signal was excited at 633 nm. Our gating strategy assured that the same cell population in terms of size and granularity was counted in each condition. In each set of experiments, untransfected or unlabeled cells, as well as single-color controls, were used to adjust threshold values, and these settings were then used when analyzing all samples.

SARS-CoV-2-derived peptides-loaded MHC class I tetramers were generated by QuickSwitch^TM Quant HLA-A*24:02 Tetramer Kit-PE (MBL International Corporation, Cat# TB-7302-K1) according to the manufacturer’s protocol. The rate of peptide exchange was quantitated by flow cytometry, and the tetramers, at a rate of more than 90%, were used for staining PBMCs. After treatment with a protein kinase inhibitor, Dasatinib (Cat# A10290-25, AdooQ, 50 nM) for 30 min at 37 °C, PBMCs were stained with tetramers for 30 min on ice. After tetramer staining, cells were counterstained with anti-PE unconjugated mAb (PE001, Biolegend, 1/10 dilution) for 20 min on ice and surface stained with the following antibodies: CD3 BV421 (UCHT1, 1/50 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/Cy5.5 (HIB19, 1/100 dilution; Biolegend) was performed. Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation for 20 min on ice, the cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and the levels of tetramer⁺CD8⁺ T cells were analyzed by flow cytometry using a FACS Canto II (BD Biosciences). The data obtained by flow cytometry were analyzed with FACS Diva v9.0 (BD) and FlowJo software v10 (Tree Star).