Measuring P-STAT-5, Bcl-2, and cell survival in CD8⁺ T-cell subsets in response to IL-7 in elderly and young subjects. PBMCs from healthy elderly and young subjects were stained with antibodies to CD8, CD45RA, and CCR7. (A-C) Cells were incubated for 10 minutes with recombinant human IL-7 (10 pg/mL∼10 ng/mL) or PBS. P-STAT5 in naive, CM, EM, and EM_CD45RA+ CD8⁺ T-cell subsets was measured using flow cytometry. (A) Representative histograms of P-STAT5 (shaded) and isotype control (open) staining. (B) Correlation between the frequencies of IL-7Rα^high cells and P-STAT5^high cells. (C) MDFI of P-STAT5 staining is compared between elderly (○, n = 10) and young (•, n = 10) subjects. (D-E) Cells stained with antibodies to CD8, CD45RA, and CCR7 were sorted into naive, CM, EM, and EM_CD45RA+ CD8⁺ T cells using a FACSAria. Cells were incubated for 6 days in the presence of recombinant human IL-7 (100 ng/mL) or PBS. (D) Cells were permeabilized and stained with antibodies to Bcl-2 (representative data from 4 separate subjects in each group). (E) Cells were stained with annexin V and 7-AAD to identify live cells (annexin V^- and 7-AAD^-). The difference in the frequency of live cells between samples treated with IL-7 and PBS was calculated from each sample (○ indicates elderly; •, young). P values were obtained with the Pearson correlation (B) or the Mann-Whitney U test (C,E).