Cd69 fn50

Human single cell suspensions were surface stained with fluorescence conjugated mAb to CD134 (L106, BD Bioscience), CD137 (4B4-1, BD Bioscience), CD69 (FN50, eBioscience), CD56 (N901, Beckman Coulter), CD3 (SK7, eBioscience) and CD5 (OKT1, in house). Murine cells were first FcγR-blocked (2.4G2, in-house) and then stained with CD49b (DX5, eBioscience), NKp46 (29A1.4, eBioscience), CD3 (17A2, eBioscience), CD4 (RM4–5, eBioscience), CD8 (53–6.7, eBioscience) or CD25 (PC61.5, eBioscience). For intracellular FOXP3 (NRRF-30, eBioscience) staining, cells were fixed and permeabilised as per manufacturer’s protocol (eBioscience). Acquisition was performed using FACSCalibur (BD Biosciences) and analysed using Cytobank (Cytobank).

Panels of fluorochrome-labeled monoclonal antibodies (mAbs) that have been shown to be cross-reactive with AGMs, were used to label fresh whole blood and LN cells and were purchased from BD Biosciences unless otherwise stated: CD3 (SP34-2), CD4 (L200), CD8 (Sk1), CD20 (2H7, ebioscience), HLA-DR (L243), CD16 (3G8), NKG2A (Z199, Coulter), CD107a (H4A3), CXCR3 (1C6), CD69 (FN50, ebioscience), CD123 (7G3), BDCA-2 (AC144, Miltenyi), CD86 (FUN-1), CD40 (HB14, Caltag), CCR7 (3D12, ebioscience), CD11c (S-HCL-3), CD80 (L307.4), IFN-γ (45-15, Miltenyi), CD45 (D058-1283), Ki-67 (MIB-1, Dako) as well as isotype controls. FcR Blocking Reagent (Miltenyi) was used to block unwanted binding of antibodies and increase the staining specificity of cell surface antigens. For detection of IFN-γ and CD107a, cells were pre-incubated for 4 hours with brefeldin A and monensin at 37°C prior to labeling with surface-binding antibodies and then fixed and permeabilized prior to incubation with IFN-γ antibody. Cells were run on a BD LSR-II flow cytometer system, collected with BD FACS Diva 6.0 software, and analyzed with FlowJo 8.8.7 (TreeStar).