Anti p stat3 pe

Expression of cytokines and transcription factors was assessed by intracellular staining. The following antibodies were used for intracellular staining of mouse cells: anti-CD4- peridinin chlorophyll or -fluorescein isothiocyanate, anti-CD25-eFluor 450, anti-IL-17-phycoerythrin (PE), anti-Foxp3-allophycocyanin (APC), anti-IFNγ-peridinin chlorophyll-cyanine 5.5, and anti-IL-4-PE-cyanine 7 (all from eBioscience, San Diego, CA, USA). Cells were stimulated for 4 h with phorbol 12-myristate 13-acetate (Sigma) and ionomycin (Sigma) with the addition of GolgiStop (BD Bioscience). Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer's protocol. To examine the expression of phosphorylated STAT (p-STAT)3 and p-STAT5, cultured cells were stimulated with IL-6 for 30 min before harvesting and were stained with anti-p-STAT3-PE and anti-p-STAT5-Alexa Fluor 488 (both from BD Bioscience). Appropriate isotype controls were used for gate setting. Cells were analyzed on fluorescence-activated cell sorting LSRII Fortessa, and the data were analyzed with FlowJo software version 7.6 (Tree star, Ashland, OR, USA). The experiments were conducted at least three times. The experiments were performed with individual samples from separate experiments and not using different wells from the same culture plate.