Aged MSCs accumulate mitochondria with low membrane potential, resulting in a senescent phenotype. BM was isolated from young and aged C57Bl/6 mice. (A) Gating strategy for MSCs. (B) MSC counts per 100 000 BM cells from young and aged mice. (C) Representative flow plot of TMRM^hi and TMRM low MSC populations in young (blue) and aged (red) mice and comparison of TMRM^hi MSC frequency. (D) Mean fluorescent index (MFI) of MTG in MSCs from young and aged C57Bl/6 mice was measured by using flow cytometry, and mitochondrial DNA content was measured relative to genomic DNA by using TaqMan PCR. (E) MSCs were separated by FACS, and gene expression of p16 and p21 was measured by RT-qPCR; expression relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown. (F) Schematic of p16-tdTom mouse model in which p16^INK4a promotor is linked to tdTom, allowing detection of cells expressing p16 by flow cytometry. (G) Representative flow plot of tdTom expression in young and aged mice and comparison of MFI. (H) Gene expression of IL-6, relative to GAPDH, in MSCs from young and aged mice separated by FACS. (I) Gene expression of laminin B1, relative to GAPDH, in MSCs from young and aged mice separated by FACS. (J) Gene expression of p16 and p21, relative to GAPDH, in LSKs separated by FACS. ∗P < .05, ∗∗P < .01 using the Mann-Whitney U test or two-way analysis of variance. FSC, forward scatter; ns, not significant; SSC, side scatter.