Fluorescein isothiocyanate fitc conjugated anti cd4

Non-adherent lymphocytes or purified CD4⁺ T cells were collected at 0, 1, 2 and 3 days following stimulation, and the cells were stained with phycoerythrin (PE)-conjugated anti-CD83, fluorescein isothiocyanate (FITC)-conjugated anti-CD4 or FITC-conjugated anti-CD25 (All from BioLegend, San Diego, CA, USA) and incubated on ice for 30 min in the dark. The controls were prepared using FITC- or PE-conjugated isotype controls. The intracellular staining of TGF-β-treated CD4⁺ T cells with anti-Foxp3 mAb was performed using an anti-human Foxp3 staining set (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. The cells were subsequently analyzed using a flow cytometer (Beckman Coulter Epics XL; Beckman Coulter, Miami, FL, USA) by using a two-color acquisition method and the data were analyzed by using FlowJo 7.6.1 software (Treestar, Inc., Ashland, OR, USA).