Data were generated as described elsewhere (9 (link), 16 (link)–19 (link)). Spontaneous
cell death was evaluated in fresh samples using 40 nM DIOC6 (Invitrogen), as
described previously (16 (link)), combined with
propidium iodide (Sigma-Aldrich, Madrid, Spain). For other staining panels flow
cytometry was performed using a series of different antibody combinations. In
brief, T-cell maturation and immunosenescence were evaluated using CD3-APC-Cy7,
CD4-PerCP-Cy5.5, CD8-V500, CD57-FITC, CD27-APC, CD28-PE, CCR7-PE-Cy7, and
CD45RA-V450 (BD Bioscience). Proliferation and Treg phenotype were assessed
using an antibody panel comprising Ki67-FITC, FOXP3-PE, CD25- PE-Cy7,
CD127-Alexa Fluor 647, CD3-APC-Cy7, CD4-V450, and CD8-V500. The activation panel
included CD95-FITC, CD38-PE, HLA-DR-PerCP, CD3-APCCy7, CD4-APC, and CD8-PE-Cy7.
All the stained cells were collected in LSRII flow cytometer (Becton Dickinson)
and analyzed with FlowJo Software (version 7.6.5).
cell death was evaluated in fresh samples using 40 nM DIOC6 (Invitrogen), as
described previously (16 (link)), combined with
propidium iodide (Sigma-Aldrich, Madrid, Spain). For other staining panels flow
cytometry was performed using a series of different antibody combinations. In
brief, T-cell maturation and immunosenescence were evaluated using CD3-APC-Cy7,
CD4-PerCP-Cy5.5, CD8-V500, CD57-FITC, CD27-APC, CD28-PE, CCR7-PE-Cy7, and
CD45RA-V450 (BD Bioscience). Proliferation and Treg phenotype were assessed
using an antibody panel comprising Ki67-FITC, FOXP3-PE, CD25- PE-Cy7,
CD127-Alexa Fluor 647, CD3-APC-Cy7, CD4-V450, and CD8-V500. The activation panel
included CD95-FITC, CD38-PE, HLA-DR-PerCP, CD3-APCCy7, CD4-APC, and CD8-PE-Cy7.
All the stained cells were collected in LSRII flow cytometer (Becton Dickinson)
and analyzed with FlowJo Software (version 7.6.5).
