Pe cy7 conjugated anti cd45ra

For analyses of the phenotype and frequency of each cell subset, peridin chlorophyll protein (PerCP)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD45RO, fluorescein isothiocyanate (FITC)-conjugated anti-interferon (IFN)-γ, allophycocyanin (APC)-conjugated anti-IL-17a, PE-conjugated anti-IL-23R, FITC-conjugated anti-CD4, APC-conjugated anti-CD25, PE-conjugated anti-FoxP3, APC-conjugated anti-CD39 (all from eBioscience, American) and phycoerythrin cyanin-7 (PE-Cy7)-conjugated anti-CD45RA (BD Bioscience) were used. For intracellular IL-17a and IFN-γ staining, cells were stimulated with 50 ng/mL phorbol myristate acetate (PMA) and 1 μg/mL ionomycin (both from Sigma-Aldrich) in the presence of 10 μg/mL GolgiStop (BD Bioscience) for 5 hours at 37 °C in a humidified 5% CO₂ incubator before staining. The cells were extracellularly stained, then treated with Cytofix/Cytoperm solution and Perm/Wash solution (BD Bioscience). For intranuclear FoxP3 staining, cells were first extracellularly stained and then treated with Fixation/Permeabilization and Permeabilization Buffer (FoxP3 Staining Buffer Set, eBioscience, American). The appropriate isotype controls were used in all the staining procedures, and the stained cells were processed with a BD FACS Calibur and analysed using FlowJo software, version 7.6.1.

Multi-color flow cytometry of PBMCs was performed at CNUH. The following human antibodies were used for multi-color flow cytometry: Brilliant Violet 421-conjugated anti-CD25, PE-Cy7 conjugated anti-CD45RA, and APC-Cy7 conjugated anti-CD3 (all from BD Biosciences, San Jose, CA, USA) and Brilliant Violet 605-conjugated anti-CD8, PerCP-Cy5.5-conjugated anti-CD4, FITC-conjugated anti-PD-1, and APC-conjugated anti-CD127 (all from BioLegend, San Diego, CA, USA). For intracellular staining of FOXP3, cells were fixed and permeabilized after surface staining using the Foxp3/Transcription factor staining buffer set (eBioscience) and incubated with PE-conjugated FOXP3 antibody (eBioscience, San Diego, CA, USA). To exclude dead cells, single-cell suspensions were first incubated for 20 min in viability dye (LIVE/DEAD Fixable Aqua, Thermo Fisher). Stained cells were analyzed using BD LSR Fortessa X-20 flow cytometry (BD Biosciences). Fluorescence-activated cell sorting (FACS) analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).