Cd95 apc

UCLA Immune Assessment Core performed the analysis of immunosenescent cells for 144 whole blood samples, as described previously [71 (link)]. Briefly, Total CD3 + T-cells, CD4 + T-cells, CD8 + T-cells, CD19 + B-cells, and CD56 + /CD16 + NK-cells were enumerated in EDTA whole blood with the BD Multitest 6-color TBNK reagent and BD Trucount tubes following the manufacturer’s instructions, acquired on a BD FACSCanto II and analyzed with the BD FACSCanto Software. CD8 + T-cell sub setting was performed by staining 50 μl of EDTA whole blood with CD3 FITC, CD8 PerCP, CD28 PE, and CD95 APC (BD) for 10 min, followed by BD FACS Lysing used according to the manufacturer’s instructions. At least 10,000 lymphocyte events per sample were acquired and analyzed using DIVA 8.0 software on BD FACSCanto II.

Cryopreserved PBMCs collected at T0 and T12 were thawed and stained (1x10⁶ cells) with fluorochrome-labelled antibodies for the flow cytometric study of lymphocyte surface phenotypes. To check cell viability, cells were stained with 7-aminoactynomycin D (7-AAD, BD Biosciences, San Jose, California, USA) for 30 min in the dark at 4°C. Only samples with cellular viability greater than 70% were used for experiments.
The following antibodies were used: HLA-DR-FITC, CD38-PE, CCR7-PeCy7, CD45RA-PeCy5, CD27-PE, CD95-APC, α4β7integrin-APC CCR6-PeCy7, CD161-APC (BD Biosciences, San Jose, California, USA), CCR9-FITC (R&D Systems, Minneapolis, MN, USA).
We evaluated CD4+ and CD8+ activation (HLA-DR+CD38+), maturation (naïve: CCR7+CD45RA+; central memory: CCR7+CD45RA-; effector memory: CCR7-CD45RA-; terminally differentiated: CCR7-CD45RA+) and stem cell-like memory T cells (Tscm; CCR7+CD45RA+CD27+CD95+). CD4+ T-cell populations involved in mucosal immunity (CCR9+α4β7+; CCR6+CD161+) were also studied.
Cells were run on a FACS VERSE cytometer (BD Biosciences, San Jose, California, USA).

Expression of surface markers associated with mesenchymal stem cells, such as CD13, CD34, CD44, CD45, CD95, CD105, HLA-DR and HLA-ABC, was evaluated by flow cytometry. Cultured cells at passage number 9 were detached from dishes with nonenzymatic cell dissociation solution (Sigma) and washed twice in PBS at room temperature. Cell pellets containing 2.5 × 10⁵ cells were resuspended in 0.1 mM PBS and then incubated with isotype control IgG or specific antibodies, including CD-34, CD-44 and 90-PE (BD Biosciences, San Jose, CA, USA), CD-95-APC (BD Biosciences), CD-45-FITC, HLA-ABC-FITC and HLA-DR-FITC (BD Biosciences). A total of 1 × 10⁴ cells were analyzed using a FACScan flow cytometer (BD Biosciences) and quantified with CellQuest Pro Software (BD Biosciences).