Anti cd4 apc efluor 780 sk3

The following antibodies were used for Phosflow and IL-6R phenotype analysis in samples processed at Newcastle: anti-CD3-Pacific blue (UCHT1), anti-CD19-FITC (4G7), anti-CD19-APC (HIB19), anti-Stat3 (pY705)-Alexa Fluor 647 (4/P-STAT3) and anti-Stat1 (pY701)-Alexa Fluor 647 (4a; all BD Biosciences, Oxford, UK); anti-CD4-APC-eFluor 780 (SK3; eBioscience, Hatfield, UK); IL-6R-Fluorescein (17506; R&D Systems Europe, Abingdon, UK). Phosflow was performed on whole blood, which was either left unstimulated or stimulated with 100 ng/mL IL-6 (PeproTech EC, London, UK) for 15 min at 37°C. BD Phosflow Lyse/Fix and BD Phosflow Perm Buffer III (both BD Biosciences) were used as per the manufacturers’ instructions. IL-6R expression was assessed in whole blood using BD FACS Lysing Solution (BD Biosciences) as per the manufacturers’ instructions. Data were collected on a BD FACSCanto II (BD Biosciences) and analysed using FlowJo (Treestar, Ashland, Oregon, USA). The protocol followed for samples processed in Brisbane was similar, using anti-CD3-Pacific blue (UCHT1), and anti-Stat3 (pY705)-PE (4/P-STAT3; BD Biosciences), with a Gallios flow cytometer and Kaluza software for data acquisition/analysis (both Beckman Coulter). Flow-Set Pro Fluorospheres (Beckman Coulter) were used to normalise median fluorescence intensities (MFI) for pSTAT3 measurements between data sets.