Cd45ro pe

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.

Cells were stained for 30 min at 4°C in PBS containing 0.01% NaN₃ and 0.5% BSA with directly labeled antibodies against: CXCR3 Alexa fluor488, CCR5 PE, CCR4 PerCP‐Cy5.5, CCR7 PE‐Cy7, CCR6 Alexa647, CD4 APC‐H7, CD3 V500, CD69 PerCP, CD45 V500 (all from BD Biosciences); CD3 FITC (Sanquin); CD4 PE‐Cy7, CD45 RA eFluor450, and CD45RO PE (all from eBioscience Inc., San Diego, CA). For intracellular cytokine staining, we used IL‐10 PE‐Cy7 (BioLegend, San Diego, CA, USA) and the Th1/Th2/Th17 kit from BD Biosciences. For intracellular staining, after cell surface staining, cells were washed and fixed with FACS Cytofix fixation buffer (BD Biosciences). Following permeabilization (FACS Perm/Wash buffer [BD Biosciences]) cells were stained with markers as indicated. Cells were analyzed on a FACS Canto II (BD Biosciences). CS&T beads were run daily and the same machine with dedicated cytometer configuration was used for the measurements of the samples throughout the study with regular control runs to check compensation settings. Data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI for cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest were normalized to the gMFI of the cytokine negative population.