Brilliant violet 421 conjugated anti cd25

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).

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 (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 (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 a PE-conjugated FOXP3 antibody (eBioscience, San Diego, CA, USA). To exclude dead cells, single-cell suspensions were first incubated for 20 min in a viability dye (LIVE/DEAD Fixable Aqua, Thermo Fisher). The stained cells were analyzed using BD LSR Fortessa X-20 flow cytometry (BD Biosciences). Fluorescence-activated cell sorting was performed using FlowJo software (Tree Star, Ashland, OR, USA).