Pe anti human cd25 antibody

The cells were collected and incubated with the corresponding antibodies (PE/Cyanine7 anti-human CD147 antibody, 306,216, BioLegend; FITC anti-human CD3 antibody, 300,406, BioLegend; APC/Cyanine7 anti-human CD4 antibody, 317,418, BioLegend; PerCP/Cyanine5.5 anti-human CD8a antibody, 301,032, BioLegend; FITC-Labeled Recombinant Protein L, RPL-PF141, ACRO Biosystems; PE anti-human CD25 antibody, 302,606, BioLegend; APC anti-human CD69 antibody, 310,910, BioLegend; APC anti-human CD107a antibody, 328,619, BioLegend; PE/Cyanine7 anti-human PD-1 antibody, 329,917, BioLegend; PerCP/Cyanine5.5 anti-human CD8a antibody, 301,032, BioLegend; PE-anti human IL-2 antibody, 500,307, BioLegend; PE/Cyanine7 anti-human IFN-γ antibody, 502,528, BioLegend; APC anti-human TNF-α antibody, 502,912, BioLegend) at 4 °C for 30 min. After washed with PBS, all samples were detected by FACS Aria instrument (BD Biosciences) and data were analyzed with FlowJo7.6 software (Tree Star).

Harvested CD4⁺ T cells were counted for Th17 and Treg percentages using flow cytometry (FACScalibur). Before detection of Th17 cells, cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 1 µg/ml Ionomycin (Sigma-Aldrich) in the presence of Brefeldin A (BD Pharmingen) at 37°C for 4 hours. Cells were stained with FITC anti-human CD4 antibody (Biolegend). Afterwards, cells were fixed, permeabilized, and labelled with and PerCP/Cy5.5 anti-human IL-17A (Biolegend). Th17 were cells which expressed CD4⁺ IL-17A⁺. For detection of Treg, cells were labelled with PerCP anti-human CD4 antibody (Biolegend) and PE anti-human CD25 antibody (Biolegend). FITC anti-human FoxP3 antibody (Biolegend) was added later after cells were fixed and permeabilized. Treg were cells which expressed CD4⁺ CD25⁺ FoxP3⁺.