Clone 4 p stat3

For analysis of STAT phosphorylation, thawed PBMCs were rested in serum-free X-VIVO-15 medium for 1 hour, washed with phosphate buffered saline (PBS), and stimulated at 10⁶ cells per100 μl X-VIVO-15 with recombinant human IL-6 (rhIL-6) (2 ng/ml) (BD, catalog no. 550071) for 10 min, rhIL-10 (5 ng/ml) (BD, catalog no. 554611)for 20 min) or rhIL-27 (10 ng/ml) (eBioscience, catalog no. 14-8279) for 20 min. Cells were fixed and permeabilized using Fix Buffer I and Perm Buffer III (BD), respectively. Subsequently, cells were stained simultaneously for CD4 (BD, clone SK3), CD45RA (BD, clone HI100), CD8 (Beckman Coulter, clone SFCl121Thy2D3), pSTAT3 pY705 (BD, clone 4/P-STAT3), and pSTAT1 pY701 (BD, clone 4a) and incubated at room temperature for 45 min. Cell surface staining for IL-6R (BD, clone M5), gp130 (BD, clone AM64), CCR7 (BioLegend, clone G043H7), CD62L (BioLegend, clone DREG-56), CXCR6 (BioLegend, clone K041E5), CCR5 (BD, clone 2D7/CCR5), CCR2 (BD, clone 48607), ADAM17 (R&D Systems, clone #111633) and ADAM10 (BioLegend, clone SHM14) was carried out without previous cell fixation. Cells were acquired on a BD FACSCanto II and data were analyzed using FlowJo version vX.06 (Tree Star).

For analysis of STAT3 expression and STAT3 phosphorylation, PBMCs were used either unstimulated or stimulated with human recombinant IL-6 (0.5μg/ml, PeproTech, Hamburg, Germany) for 15 min at 37°C. EBV-transformed B cell lines were stimulated with IFN-α (0.5μg/ml, PeproTech, Hamburg, Germany) for 5, 15, 60 and 150 min at 37°C. Cells were fixed (Lyse/Fix buffer, BD Biosciences) and permeabilized (Fix/Perm III buffer, BD Biosciences) according to the manufacturer΄s instructions. Cells were stained with anti-STAT3-FITC (clone #232209; R&D Systems Wiesbaden-Nordenstadt, Germany), anti CD19 BV421 (clone HIB19, Biolegend), or with phospho-specific PE-coupled anti-STAT3 (pY705) antibodies (clone 4/P-STAT3, BD Biosciences), CD3-FITC (clone SK7, BD Bioscience). Fixable viability dye eFluor 506 (eBioscience, Frankfurt, Germany) was used according to the manufacturer΄s instructions.