Pe conjugated anti foxp3 mab

Peripheral blood samples (150 µL) were incubated with APC-conjugated anti-CD3 (3.5 µL), PerCP-conjugated anti-CD4 (3.5 µL) or PerCP-conjugated anti-CD8 (3.5 µL) and FITC-conjugated anti-CD25 (3.5 µL) monoclonal antibodies (mAbs) (BD Biosciences) at 4°C for 20 min. RBC lysis buffer (1 mL) (Boster Biosciences) was then added, and the samples were incubated for another 10 min in the dark. The tubes were centrifuged at 200×g for 5 min. The supernatants were discarded, and the cells were washed in PBS. The cells were resuspended in Foxp3 fixation/permeabilization concentrate and diluent solutions (500 µL) (eBioscience) and incubated at room temperature in the dark for 30 min. The tubes were centrifuged at 200×g for 5 min, the supernatants were discarded, and the cells were then washed twice in PBS. The cells were incubated with PE-conjugated anti-FoxP3 mAb (7 µL) (eBioscience) at 4°C for 30 min. Isotype-matched mAbs were used as negative controls during each staining stage. After washing with PBS, the stained cells were analyzed using flow cytometry with a FACS Calibur (BD Biosciences) and analyzed with FCS Express V3 software (De Novo Software).

Single cells were incubated with corresponding membrane monoclonal antibodies (mAbs) against FITC-conjugated anti-CD4 (Cat# 88-8111-40; eBioscience, USA) and APC-conjugated anti-CD25 (Cat# 88-8111-40; eBioscience, USA) at 4°C in the dark for 30 min, and then washed once with cold PBS. Subsequently, cells were fixed and permeabilized according to the manufacturer’s instructions using a fixation and permeabilization kit (Cat# 88-8111-40; eBioscience, USA) and then washed with PBS. Then, cells were then stained with a PE-conjugated anti-Foxp3 mAb (Cat# 88-8111-40; eBioscience, USA) at 4°C for 45 min in the dark. Finally, after being washed twice with PBS, cells were resuspended in 500 μL of PBS and further used for flow cytometry analysis (Cytometer LX, Beckman, USA).