Pe cy5 conjugated anti humancd25

Flow cytometry was used to measure the proportion of Th17 cells and
CD4⁺CD25⁺Treg cells in mononuclear cells in peripheral
blood. For Tregs detection, lymphocytes were stained with fluorescein
isothiocyanate (FITC)-conjugated anti-human CD4, PE-Cy5-conjugated anti-human
CD25, and PE-conjugated anti-human Foxp3 mAbs (eBioscience, San Diego, CA, USA)
and the proportion of CD25⁺Foxp3⁺ lymphocytes gated in
CD4⁺ lymphocytes were defined. For the detection of Th17 cells,
single-cell suspensions were stimulated with 50 ng/mL phorbol myristate acetate,
1 µg/mL ionomycin, and 2 µg/mL monensin. After 5 h, cells were stained with
FITC-conjugated anti-human CD3 and PE-conjugated anti-human CD4 mAbs, fixed,
permeabilized, and stained with PE-Cy5-conjugated anti-human IL-17A mAb
according to the intracellular staining kit (Invitrogen, Carlsbad, CA, USA)
instructions. We determined the proportion of CD4 + IL-17 + lymphocytes gated in
mononuclear cells in peripheral blood.

For detection of Th17 cells, PBMCs (2 × 10⁶ cells/ml) were stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) containing ionomycin (1 μμ) and Monensin (500 ng/ml). Upon harvesting, the cells were stained for CD4 by incubating the cells with fluorescein isothiocyanate (FITC)-conjugated antihuman CD4 (BioLegend) at 4°C for 30 min. After fixation and permeabilization, the cells were further stained with phycoerythrin (PE)-conjugated antihuman IL-17A (eBioscience, San Diego, CA, USA) for detection of Th17 cells. For detection of Treg cells, PBMCs were incubated with allophycocyanin (APC)-conjugated antihuman CD4, PE-Cy5-conjugated antihuman CD25, and FITC-conjugated antihuman CD127 (eBioscience) in the dark for 30 min at room temperature. Finally, the cells were analyzed by multicolor flow cytometry on a FACSCalibur (Becton Dickinson).