Fluorochrome conjugated monoclonal antibodies specific for human cd4 fitc and cd25 pe

For the CD4⁺ cells or PBMCs analysis, harvested cells were re-suspended in 50 μL of fluorescent-activated cell sorting buffer and stained for 30 min on ice for respective membrane antigens, using fluorochrome-conjugated monoclonal antibodies specific for human CD4–FITC and CD25-PE (both from (BD Pharmingen, San Diego, CA, USA). Cells were fixed and permeabilised in the next step using the FoxP3/transcription factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, intracellular staining using anti FoxP3-APC antibody were performed. After the washing, cells were acquired and analysed using a FACS Canto cell sorter/cytometer and Diva software. Appropriate isotype controls were used in all experiments.

For the CD4⁺ cells or PBMCs analysis, harvested cells were re-suspended in 50 μL of cell sorting buffer and stained for 30 min on ice for respective membrane antigens, using fluorochrome-conjugated monoclonal antibodies specific for human CD4–FITC, and CD25-PE (both from (BD Pharmingen, San Diego, CA, USA). In the next step, cells were fixed and permeabilized using FoxP3 transcription factor Staining Buffer Set (Thermo Fisher Scientific, MA, USA). Subsequently, intracellular staining using anti-FoxP3-APC antibody was performed. After the washing step, cells were acquired and analyzed using a FACS Canto cell cytometer and Diva software. Appropriate isotype controls were used in all experiments.