For intracellular staining of p53 and cleaved caspase-3, total bone marrow cells were stained with the lineage-depletion kit (130-090-858, MACS Miltenyi Biotec) following the manufacturer’s instructions and passed through LS magnetic columns. Lineage-depleted cells were spun down for 5 min at 1,200 r.p.m. and the pellets were resuspended in 200 μl MACS buffer with the antibodies described above for HSC quantification. In parallel, 3 × 106 total bone marrow cells were stained with antibodies against committed lineages: CD45R/B220 (PE, clone RA3-6B2, BD Pharmingen) and IgM (FITC, clone II/41, BD Pharmingen) for B cell progenitors; TER-119 (FITC, clone TER-119, BD Pharmingen) and CD71 (PE, clone C2, BD Pharmingen) for erythroid maturation; and CD11b/Mac-1 (PE, clone M1/70, BD Pharmingen) and Ly-6G/Gr-1 (FITC, clone 1A8, eBioscience) for monocyte/granulocyte progenitors. After antibody staining, cells were washed, then fixed and permeabilized with BD Cytofix/Cytoperm solution (554722, BD Pharmingen) following the manufacturer’s instructions. Finally, cells were stained with either anti-p53 (AlexaFluor647, clone 1C12, Cell Signalling) or anti-cleaved-caspase-3 (AlexaFluor647, clone D3E9, Cell Signalling) antibodies.
