Anti human cd25 pe

Cell surface markers were evaluated by flow cytometry using a standardized protocol. Cells were kept on ice during all the procedures; 0.5 × 10⁶ cells were stained with anti-human CD69 FITC (eBioscience, Cat. 11-0699-42) or anti-human CD25 PE (eBioscience, Cat. 12-0259-80). Detection of cell surface markers was conducted using a Beckman-Coulter Gallios Flow Cytometer (BD Biosciences), and data were analyzed by Kaluza Analysis Software. Live/dead assays were determined using the Aqua Dead Cell Stain Kit (ThermoFisher, Cat. L34957).

Thawed PBMC were washed and re-suspended, and the cell viability was measured by Trypan blue staining. A total of 2 × 10⁶ cells were stained with anti-human CD4-FITC, anti-human CD25-PE, anti-human CD127-PE-Cy5, anti-human PD-1-APC and anti-human PD-L1-PE-Cy7 for surface antigens (eBioscience, San Diego, CA, USA), in accordance with the manufacturer’s instructions. For intracellular cytokine detection, 2 × 10⁶ cells were stimulated with 2 μL of Cell Stimulation Cocktail and 2 μL of Protein Transport Inhibitor Cocktail (eBioscience, San Diego, CA, USA) for 5 h. Collected cells were first stained with fluorescein-labelled mAbs for surface antigens, followed by fixation using IC Fixation buffer (eBioscience, San Diego, CA, USA). Then, the cells were stained with anti-human IL-17A-PE (eBioscience, San Diego, CA, USA) in an appropriate volume of 1 × Permeabilization Buffer (eBioscience, San Diego, CA, USA) for 25 min. Finally, the cells were re-suspended in 300 μL of PBS for subsequent flow cytometric analysis. All data was acquired on FACScalibur (BD Biosciences, San Jose, CA), and processed using the CellQuest program (Becton Dickinson, Franklin Lakes, NJ).