Hematopoietic stem cells exhibit p53-dependent differences in stem-cell numbers in old mice. (A) Numbers of total bone marrow cells from young and old p53^+/+, p53^+/m, p53^+/−, and p53^−/− mice were obtained from hind limbs, counted manually and shown to be similar. Results represented are the mean ± SE of 10 to 14 mice per age/genotype. (B) LT-HSCs identified as lin^−/lo, Sca-1⁺, c-kit⁺, and Flk-2⁻ were selected by whole bone marrow antibody staining and subsequent flow cytometry. The experiments were performed in triplicate; n = 10 for young mice and n = 6 for old mice. Results shown represent the mean ± SE of the experiments. An increase in LT-HSC numbers is shown with age in p53^+/+ and p53^+/− bone marrow but not in p53^+/m bone marrow. (C) Whole bone marrow was stained with Hoechst 33342 and enriched for Sca-1⁺ cells to identify the side population (SP) HSCs. SP-HSCs isolated from 12-month p53^+/m bone marrow are reduced approximately 50% in number compared with SP-HSCs isolated from 12-month p53^+/+ bone marrow. Numbers shown are for Sca-1⁺–enriched bone marrow, which enriches approximately 10-fold for SP cells.