Apc anti human cd3 monoclonal antibody

Lymphocytes in the liver were used to count Treg cells according to the method described in a previous study [21 ]. Treg cells were stained and counted using the eBioscience™ Human Regulatory T Cell Staining Kit (Cat. No. 88-8999, Thermo Fisher Scientific, Hudson, NH, USA). Briefly, a new Treg cells counting process was used as follows. The concentration of lymphocytes was adjusted to 1×10⁷/ml. A total of 100 μl hepatic lymphocytes suspension was treated with 20 μl FITC anti-human CD4⁺ monoclonal antibody, 20 μl PE anti-human CD25⁺ monoclonal antibody, 20 μl PerCP-labeled anti-human CD45⁺ monoclonal antibody, and 20 μl APC anti-human CD3⁺ monoclonal antibody (BD Biosciences, San Jose, CA, USA) and incubated for 20 min in the dark. Then, the lymphocyte suspension was incubated with 450 μl FACS lysing solution (Catalogue No. 349202, BD Biosciences, San Jose, CA, USA) at room temperature for 15 min in the dark. The lymphocyte suspension was also centrifuged at 1500 r/min and re-suspended in 2 ml phosphate buffer solution (PBS, Sigma-Aldrich), centrifuged again, and then the supernatant was discarded. Finally, the pillets were dissolved in PBS and prepared for FACS flow cytometry (BD Biosciences, San Jose, CA, USA).

Cell surface and intracellular markers expression was evaluated on the fresh whole EDTA-anti-coagulated peripheral blood according to the method described in a previous study [20 (link)] conducted in Sweden. The lymphocytes were isolated using density gradient centrifugation at 900×g at room temperature for 20 min over Lymphoprep™ (Nycomed, Oslo, Norway) according to the manufacturer’s instructions. Briefly, for the surface staining, the lymphocytes were incubated using FITC-conjugated anti-human CD4⁺ antibody (BD Biosciences, San Jose, CA, USA), PE-conjugated anti-human CD25⁺ antibody (BD Biosciences), PerCP-labeled anti-human CD127 antibody (BD Biosciences), APC anti-human CD3⁺ monoclonal antibody (BD Biosciences), and eBioscienceTM Anti-Human Foxp3 Staining St FITC (Cat. No. 71-5776, Thermo Fisher Scientific, Hudson, NH, USA) for 20 min at 4°C in the dark. Then, the lymphocytes were centrifuged (1200 r/min) for 5 min and re-suspended in 2 ml PBS. The above samples were analyzed using an FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). All flow cytometry data were analyzed using BD DIVA software 4.1 (BD Biosciences, San Jose, CA, USA). The results are presented as the percentage of CD4⁺CD25⁺ T cells, Treg CD127⁺ T cells, or Treg Foxp3⁺ cells of total lymphocytes.