Allophycocyanin apc conjugated anti human cd25 clone bc96

DC were cultured with autologous CD3⁺ T cells (1:10) for 1 day, with or without the IDO inhibitors 1-MT-D or –L (1 mM, Sigma-Aldrich), where indicated. For immunophenotype studies, tri-color immunofluorescence was performed using fluorescein isotiocyanate (FITC)-conjugated anti-human CD4 (clone RPA-T4), phycoerytrin (PE)-conjugated anti-human Foxp3 (clone 206D) and allophycocyanin (APC)-conjugated anti-human CD25 (clone BC96, Biolegend, San Diego, CA). For cell-surface staining, 1×10⁵ cells/100 µl were incubated in the dark for 20 min at 4°C with mAbs in phosPHAte-buffered saline (PBS)-1% bovine serum albumine. Subsequently, for Foxp3 intracellular staining, cells were incubated at room temperature in the dark for 20 min with fix/perm buffer followed by 15 min with perm solution and additional 30 min with the mAb. After 2 washes, samples were analyzed using BD FACSCanto™II equipment (BD Biosciences). A minimum of 10,000 events was collected in list mode on FACSDiva software. To test their suppressive activity, purified CD4⁺CD25⁺ T cells (10⁴/well) were irradiated and added to cultures consisting of CFSE-labeled CD3⁺ T cells (10⁵/well) as responders, stimulated by 1 µg/ml PHA (Sigma-Aldrich). After 5 days, cultures were analyzed using BD FACSCanto™II equipment (BD Biosciences) and the proliferation index was calculated using FCSExpress4 software.^{24 (link)}