Pe cyanine 5 labeled anti mouse cd4 antibody

After isolation, 1 × 10⁶ PBMCs were stimulated with 50 µg/L phorbol 12‐myristate, 100 µg/L ionomycin mixture, 3 mg/L brefeldin A, and 1.4 mg/L monensin mixture, respectively, for 18 hours. For cell‐surface staining, the cells were stained with phycoerythrin (PE)‒cyanine 5‒labeled anti‐mouse CD4 antibody (eBioscience, San Diego, CA). The stained cells were then incubated with a PE‐labeled anti‐mouse IL‐4 antibody (eBioscience), a flourescein isothiocyanate‒labeled anti‐mouse IFN‐γ antibody (eBioscience), and an allophycocyanin‐labeled anti‐mouse IL‐17A antibody (eBioscience), after fixation and permeabilization using a fixation/permeabilization solution kit (eBioscience) according to manufacturer's instructions. Flow cytometric data for Th1, Th2, and Th17 cells were acquired using a flow cytometer and analyzed using FlowJo software version 7.6 (FlowJo, LLC, Ashland, OR).

For cell‐surface staining, splenocytes were stained with phycoerythrin (PE)‐cyanine5‐labeled anti‐mouse CD4 antibody (eBioscience, San Diego, CA, USA) and PE‐labeled anti‐mouse CD25 antibody (eBioscience). For intracellular staining, the cells were fixed and permeabilized using a fixation/permeabilization solution kit (eBioscience), followed by incubation with a PE‐labeled anti‐mouse IL‐4 antibody (eBioscience), a flourescein isothiocyanate (FITC)‐labeled anti‐mouse IFN‐γ antibody (eBioscience), an allophycocyanin (APC)‐labeled anti‐mouse IL‐17A antibody (eBioscience), and a FITC‐labeled anti‐mouse Foxp3 antibody (eBioscience), according to manufacturer instructions. Flow cytometric data for Th1 (CD4+IL‐4+T cells), Th2 (CD4+IFN‐γ+T cells), Th17 (CD4+IL‐17A+T cells), and Treg cells (CD4+CD25+Foxp3+T cells) were analyzed by flow cytometry (FACS Calibur; BD, USA).