Paci cblue anti human cd3

Lymphoprep® (Axis-Shield PoC AS, Oslo, Norway) was used to separate the whole blood into peripheral blood mononuclear cells (PBMCs). PBMCs were stained with antibodies, including FITC anti-human TCRαβ (BD Biosciences Pharmingen, San Diego, CA, USA), PE/Cy7 anti-human CD3 (Biolegend, San Diego, CA, USA), Paci cBlue anti-human CD3 (Biolegend), PE anti-human CD4 (Biolegend), PE/Cy7 antihuman CD4 (Biolegend), PE anti-human CD8 (Biolegend), APC anti-human CD8 (Biolegend), PE antihuman CD45RO (BD Biosciences Pharmingen), PE anti-human CCR6 (BD Biosciences Pharmingen), APC anti-human CCR4 (Biolegend), and APC anti-human CXCR3 (Biolegend), in a dark place at 4℃ for 30 min.
The PBMCs were xed in intracellular staining of RORγt and intracellularly stained using Fixation/Permeabilization Concentrate (eBioscience, San Diego, CA, USA), Fixation/Permeabilization Diluent (eBioscience), and Permeabilization buffer (eBioscience) with PE anti-human RORγt antibody (BD Biosciences Pharmingen) in a dark place at 4℃ for 45 min. The Cell Analyzer EC800 (Sony Biotechnology, Tokyo, Japan) was used for ow cytometric analysis. The data analysis was performed using FlowJo 7.6.5 software (TreeStar Inc, Ashland, OR, United States).

Evaluation of capacity to produce IL-17A and IL-10 was performed. PBMCs maintained in RPMI1640 were stimulated with 50 ng/ml of phorbol-myristate acetate (PMA) (Sigma-Aldrich, St Louis, MO, USA) and 750 ng/ml of ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences Pharmingen) at 37℃ under 5% CO 2 for 4 h. PBMCs were stained with antibodies, including FITC anti-TCRαβ (BD Biosciences Pharmingen), PE/Cy7 anti-human CD4 (Biolegend), APC anti-human CD8 (Biolegend), and Paci cBlue anti-human CD3 (Biolegend), in a dark place at 4℃ for 30 min. PBMCs were xed and intracellularly stained using BD Cyto x and Perm/Wash (BD Biosciences Pharmingen) with PE anti-human IL-17A antibody (eBioscience) and PE anti-human IL-10 antibody (Biolegend) in a dark place at 4℃ for 30 min. Cell Analyzer EC800 (Sony Biotechnology) was used for ow cytometric analysis. FlowJo 7.6.5 software (TreeStar Inc, Ashland, OR, United States) was used for data analysis.
All studies were approved by the Ethics Committee of the Yokohama City University Graduate School of Medicine (number: B160804004). Informed consent was obtained from cases, parents and/or their guardians, and healthy adults for normal control.