Age-related differences between macrophages from the BMM of young vs old mice. (A) Proliferative index (calculated using FlowJo’s Proliferation Tool) of first passage macrophages (MΦ) from young or old mice on days 2, 3, and 4 after the addition of 10 mM of CellTrace CFSE Cell Proliferation Kit (after 4 days P = .0001; ANOVA, Tukey test). The data are representative of 3 independent experiments. (B) Proliferative index as in panel A of first and second passage macrophages from young or old mice 3 days after the addition of 10 mM of CellTrace CFSE Cell Proliferation Kit (P < .0001; ANOVA, Tukey test). The data are representative of 3 independent experiments. Percentage of macrophages from young (red) or old (red) mice positive for ROS (P = .02; Student t test, n = 3) (C) or γH2AX (P = .031; Student t test) by mean fluorescence intensity (MFI) (D), as measured by flow cytometry. Macrophages had been cultured for 3 days (n = 5). (E) Representative confocal images (scale bar, 10 μm) of macrophages from young (top) or old (bottom) mice stained with an antibody to translocase of the outer membrane 20 (green) to visualize mitochondria (n = 3). Nuclei are stained with DAPI (blue). Macrophages had been cultured for 3 days before staining. (F) Heatmap depicting the Pearson correlation of normalized reads per peak (ATAC-seq). Further analysis is performed on all DARs in young (green) vs old (orange) macrophages. Statistically significant DARs were defined as abs (log2[FC]) >1 with an adjusted P value <. Overall, 239 473 consensus peaks were tested, and 9446 DARs fulfilled the statistical requirements. Clustering of DARs confirmed consistency across replicates. (G) Volcano plot summarizing all DARs in young vs old macrophages. Color codes represent significantly open (red) and closed (blue) DARs in young vs old macrophages. The top 7 DARs of both directions and their annotated genes are labeled. (H) Pathway enrichment analysis of peaks with an adjusted P value ≤.05. Depicted are top 5 enriched MSigDB hallmarks for open and closed regions in young vs old macrophages. The odds ratio quantifies the strength of association between young vs old macrophages, reflecting the enrichment of pathways in open and closed chromatin of young macrophages. Visualized is the negative log¹⁰ of the P value.