Memory CD8 T cells are of intermediate density and in G₁. Splenic T cells were purified and fractionated on a Percoll density gradient. (A) Cell size (forward scatter [FSC]) and cell-cycle analysis of gated CD8 T cells in the indicated fractions were determined by flow cytometry. The horizontal bar in the right panels represents the amount of background fluorescence emitted from Hoechst-stained cells in the pyronin Y channel. The percentages of cells in the regions corresponding to G₀, G₁, and S/G₂ plus M are indicated. (B) Cell surface expression of CD44, CD62L, and Ly6C on CD8 T cells in the indicated fractions. (C) IFN-γ production and CD69 expression by intermediate- and high-density CD8 T cells stimulated with (solid line) or without (shaded histogram) PMA/Iono (left panel). (D) T cells were enriched from LCMV-immunized mice and fractionated on a Percoll density gradient. The distribution of gp33-41 tetramer-positive CD8 T cells in the intermediate- and high-density fractions is shown in the left panel, and the cells in the gated areas were analyzed for size (middle panel) and cell-cycle state (right panels). (E) Cell surface expression of the indicated markers on intermediate-density gp33-41 tetramer-positive CD8 T cells versus high-density CD8 cells. (F) IFN-γ production and CD69 expression by intermediate-density gp33-41 tetramer-positive and high-density CD8 T cells.