Cycling, turnover, and survival of TCF-1-deficient DN cells. (A) Thymocytes were stained with a lin cocktail, including CD44, for CD25, and icTCRβ. Histograms show the relative DNA content among the indicated DN subsets, as determined by Hoechst staining. Numbers represent the mean of cells in S/G₂/M phase of the cell cycle (± SD). Of note, TCF-1-deficient thymocytes yield a normal Hoechst profile and no apparent underrepresentation of cells in G₂/M phase when Hoechst staining is performed in the absence of icTCRβ staining (data not shown). (B) Mice were injected intraperitoneally with the nucleotide analog BrdU and put on drinking water containing BrdU. Thymocytes were collected at the indicated time points and stained with antibodies to CD4, CD8, TCRβ, and BrdU or the lineage cocktail (including CD44), CD25, icTCRβ, and BrdU. The graph depicts the mean percentage (± SD) of BrdU⁺ cells in the indicated thymocyte subset. (C) Total thymocytes were incubated for the indicated periods of time in complete culture medium (without the addition of growth factors). After collection they were stained with antibodies to the lineage cocktail, CD44 and CD25. For each time point, the presence of viable DN3 or DN4 was estimated relative to a fixed number of added microspheres. The ratio of DN3 or (DN4) to beads at the onset of the culture was set to 100%. The graphs show the mean percentage (± SD) of surviving cells relative to the onset of the culture. (D) Thymocytes were surface stained for CD25, the lin cocktail (including CD44), and annexin V. After fixation and permeabilization, thymocytes were stained for icTCRβ. Histograms show the mean percentage of annexin V-positive cells (± SD) in each of the indicated DN subset.