To identify NK and CD8+ T cells subsets, Blood samples (about 10 ml) were
collected in EDTA-containing tubes. The peripheral blood mononuclear cells (PBMCs) were
first isolated using Ficoll-Hypaque (Innotrain, Germany) density gradient centrifugation
(1000×g at room temperature) and then washed two times with phosphate-buffered saline
(PBS, Euroimmun, Germany). Centrifugation of samples was at 300 ×g and 4°C. Subsequently,
isolated PBMCs (106 cells/100 µl) were stained with anti-CD8-APC, CD45RO-PE,
CD27-Percp-eFlour780, CD28-Pe-Cy7, CD57-FITC, and CD56-PerCp-eFlour710 monoclonal
antibodies (mAbs) (all from eBioscience, USA) and incubated at 4°C for 30 minutes. The
samples were fixed with formaldehyde and analyzed within 24 hours by flow cytometer (BD
FACSAria, USA). At least 50,000 events were counted for each sample.
collected in EDTA-containing tubes. The peripheral blood mononuclear cells (PBMCs) were
first isolated using Ficoll-Hypaque (Innotrain, Germany) density gradient centrifugation
(1000×g at room temperature) and then washed two times with phosphate-buffered saline
(PBS, Euroimmun, Germany). Centrifugation of samples was at 300 ×g and 4°C. Subsequently,
isolated PBMCs (106 cells/100 µl) were stained with anti-CD8-APC, CD45RO-PE,
CD27-Percp-eFlour780, CD28-Pe-Cy7, CD57-FITC, and CD56-PerCp-eFlour710 monoclonal
antibodies (mAbs) (all from eBioscience, USA) and incubated at 4°C for 30 minutes. The
samples were fixed with formaldehyde and analyzed within 24 hours by flow cytometer (BD
FACSAria, USA). At least 50,000 events were counted for each sample.