Apc conjugated anti human mouse p stat5 antibody

PBMCs were isolated by density gradient sedimentation of whole blood before being resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Biosera, France) supplemented with 10% fetal bovine serum (FBS) (Biosera, France) and 1% penicillin/streptomycin (Biosera, France). Subsequently, the PBMCs (5 × 10⁵ cells/ml) were stimulated with IL-2 (100 ng/ml) for 15 min at 37 °C in 5% CO2. The cells were washed with PBS and then stained with a FITC-conjugated anti-human CD4 antibody. After surface staining, 4% formaldehyde was used to fix the cells in the dark at room temperature for 15 min. The fixed cells were washed twice with PBS and then permeabilized with ice-cold methanol for 10 min at 4 °C. Intracellular staining was performed with an APC-conjugated anti-human/mouse p-STAT5 antibody (eBioscience, USA) for 1 h at 4 °C, after which the stained cells were washed three times with PBS. The percentages of stained cells were measured using a Cytoflex Lx flow cytometer (Beckman, USA) and analyzed by CytExpert version 2.3.0.84 software.

Splenocytes were obtained from mice in each group by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry. The percentage of Tregs in the splenocyte population was determined via flow cytometry according to the methods used for human specimens mentioned above. Tregs were sorted from mouse spleens using a CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Germany) according to the reagent manufacturer's instructions [[34] (link), [35] (link), [36] (link), [37] (link), [38] (link), [39] (link)]. Partial Tregs were fixed and permeabilized with a Transcription Factor Staining Buffer Set (eBioscience, USA) and then stained with an anti-BLIMP-1 mAb (Cell Signaling Technology, USA) or anti-IRF4 mAb (Cell Signaling Technology, USA). The labeled cells were examined on a Cytoflex Lx flow cytometer and analyzed using CytExpert version 2.3.0.84 software. The other Tregs were stimulated with IL-2 (100 ng/ml) for 15 min at 37 °C in 5% CO2 before staining with an APC-conjugated anti-human/mouse p-STAT5 antibody (eBioscience, USA). Finally, the level of p-STAT5 in Tregs was measured using a Cytoflex Lx flow cytometer (Beckman, USA) and analyzed with CytExpert version 2.3.0.84 software.