MHC‐I tetramer targeting the immunodominant epitope of the influenza nucleoprotein (DbNP366–374 – ASNENMETM, DbPA224–233 – SSLENFRAYV) was produced in‐house and conjugated to streptavidin‐APC/PE (Life Technologies Australia Pty Ltd) at a 1:250 dilution at room temperature for 1h. Cells were stained with combinations of fluorochrome‐conjugated antibodies: PerCP‐Cy5.5‐CD3 (#551163), PE‐CD8 (#561095), BV421‐I‐Ab (#562928), APC‐CD44 (#553133), FITC‐CD44 (#553133), BV711‐CD38 (#740697), PerCP‐Cy5.5‐CD8 (#551162), APC/eF780‐CD62L (#47‐0621‐82), APC‐CD11c (#17011481), PE‐Cy7‐TCR‐va2 (#560624) from BD Biosciences, USA; and PE‐Cy7‐CD38 (#102718), BV785‐PD‐1 (#329908), APC‐Cy7‐CD45.1 (#110716), FITC‐I‐Ab (#116406), Pacific Blue‐I‐Ab (#116422), FITC‐CD19 (#115506) from Biolegend, USA. AF700‐CD3 (#56003382) was purchased from Invitrogen, USA.
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).