Cells were labeled with fluorescent antibodies against the following targets: CD8, CCR7 (clone 150503), CD95 (clone DX2), CD122 (clone Mik-β2), T-BET (clone O4-46), GATA3 (clone L50-823), IFN-γ, CD45RO (clone 4S.B3, all from BD Pharmingen), CD62L, and CD45RA (clone 2H4; Beckman coulter, Miami, FL).
Single-cell suspensions were prepared. The cell surface was stained for 30 minutes at 4°C with anti-CD8, anti-CCR7, anti-CD95, anti-CD122, anti-CD45RO, anti-CD62L, or anti-CD45RA.
For intracellular cytotoxic cytokine staining, cells were stimulated with 10 μg/mL staphylococcal enterotoxin B (Sigma-Aldrich, St. Louis, MO), 100 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich), and 1 μg/mL anti-human CD28 (R&D) for 6 hours, followed by addition of brefeldin A (5 μg/mL, Sigma-Aldrich) at 2 hours before detection. Cells were washed twice in phosphate buffered saline (PBS), fixed and permeabilized with Cytofix/Cytoperm and Fixation/Permeabilization Solution Kit (BD Cytoperm). Stained samples were analyzed in a BD LSR II Fortessa and flow cytometric data were analyzed with FlowJo software (Tree Star).
Single-cell suspensions were prepared. The cell surface was stained for 30 minutes at 4°C with anti-CD8, anti-CCR7, anti-CD95, anti-CD122, anti-CD45RO, anti-CD62L, or anti-CD45RA.
For intracellular cytotoxic cytokine staining, cells were stimulated with 10 μg/mL staphylococcal enterotoxin B (Sigma-Aldrich, St. Louis, MO), 100 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich), and 1 μg/mL anti-human CD28 (R&D) for 6 hours, followed by addition of brefeldin A (5 μg/mL, Sigma-Aldrich) at 2 hours before detection. Cells were washed twice in phosphate buffered saline (PBS), fixed and permeabilized with Cytofix/Cytoperm and Fixation/Permeabilization Solution Kit (BD Cytoperm). Stained samples were analyzed in a BD LSR II Fortessa and flow cytometric data were analyzed with FlowJo software (Tree Star).