Cd45ra pe cy7

The percentage and absolute counts of naive CD4⁺ or CD8⁺ T lymphocytes; (CD3⁺CD45RA⁺CCR7⁺), central memory; (CD3⁺CD45RA⁺CCR7^neg) and effector memory (T_EM); (CD3⁺CD45RA^negCCR7^neg) were determined by flow cytometry. FITC-CD3, Pacific Blue-CD4, APCcy7-CD8, APC-CCR7, PEcy7-CD45RA were used to evaluate naive and memory T cells (BD Bioscience, San Diego, CA, USA). PE-CD56 was used to evaluate NK cells (BD Biosciences). Non-specific binding was determined using isotypic controls. Flow cytometry acquisition was performed on LSRII and analysis with Flowjo software (Treestar, Ashland, OR, USA).^{7 (link)} The percentages and absolute T-cell counts were calculated based on lymphocytes and monocytes gating.

To distinguish live and dead cells, PBMC were stained with live/dead fixable stain dye (Life technologies). After PBS washing, cells were incubated with FITC-CD3 (BD Biosciences), PerCP-Cy5.5-CD4 (BD Biosciences), BV421-CD25 (BD Biosciences), APC-CD127 (Biolegend), and PE-Cy7-CD45RA (BD Biosciences). Cells were then fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and further stained with PE-Foxp3 (BD Biosciences). Cells were analyzed with a FACSCanto II flow cytometer (BD Biosciences), and data were processed with FlowJo software (Tree Star, OR, USA).

Plasma and peripheral blood mononuclear cells (PBMC) were obtained from whole blood using lymphocyte separation medium (Corning) via density gradient centrifugation. IL-6, TNF-α, and IFN-γ were analyzed by enzyme-linked immunosorbent assay of plasma samples. For flow cytometry, PBMCs were stained with live/dead fixable stain dye (Life Technologies) to distinguish live and dead cells. After PBS washing, cells were incubated with FITC-CD3 (BD Biosciences), PerCP-Cy5.5-CD4 (BD Biosciences), BV421-CD25 (BD Biosciences), APC-CD127 (Biolegend), and PE-Cy7-CD45RA (BD Biosciences). Cells were then fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and further stained with PE-Foxp3 (BD Biosciences). As a way to consider the heterogeneity of the Treg compartment and analyze the property of Treg subgroups, we introduced the CD45RA marker to discriminate between antigen-experienced Treg (e.g., CD45RA−) and naïve Treg (e.g., CD45RA+) cells. T cells and their subpopulations were analyzed with a FACS BD LSR Fortessa™ X-20 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and data were processed with FlowJo software ver. 10 (Tree Star, Woobum, OR, USA).