CD45RA and CCR7 expression and proliferation of different CD8⁺ blood-cell subsets upon stimulation. Blood CD8⁺ cells were positively selected by magnetic sorting and labeled with an FITC-conjugated anti-CCR7 antibody and a PE-Cy5–conjugated anti-CD45RA antibody. The CD45RA⁺CCR7^– and CD45RA^–CCR7^– subsets were sorted by flow cytometry, labeled with PKh26, and distributed at approximately 3 × 10⁵ cells per well. Cells were either stimulated with beads coated with anti-CD3 and anti-CD28 antibodies in the presence of 2 U/mL IL-2 (A-C) or kept in culture with 2 U/mL IL-2 (D). Samples were collected at different time points after stimulation, and CD45RA/CCR7 surface expression was evaluated with the antibodies used for the sorting. Quadrant limits were positioned so as to include in the lower left quadrant 99% of the nonlabeled cells, and the numbers correspond to the percentage of cells in each quadrant. Proliferation was measured by the dilution of the Pkh26 dye and the dashed lines correspond to the number of divisions.