Cd3 pe cy7

Conjugated antibodies and staining reagents included MIP-1β-PE, CD3-PB, CD3-PE-Cy7, CD3-PerCP-Cy5.5, CD3-APC-Cy7, CD3-Horizon V450, CD4-PerCP-Cy5.5, CD4-AmCyan, CD4-FITC, CD8α-APC, CD8α-APC-Cy7, CD8α-AlexaFluor700, CD8α-FITC, CD8α-APC-H7, CD69- ECD (Beckman Coulter), CD20-Horizon V450, CCR7-FITC (R&D Systems), CD95-APC, CCR7-PerCP-Cy5.5, CD28-PE-Cy7 (eBioscience), granzyme B-AlexaFluor700, perforin-FITC (MabTech), IFNγ-PE-Cy7, TNFα- AlexaFluor700, IL-2-APC, CD95-PE, CD95-APC, and Aqua LIVE/DEAD Fixable Dead Cell Stain (Invitrogen). All reagents are from BD Biosciences unless indicated otherwise. For construction of monomers and tetramers, the following peptides were synthesized and purified to >95% by HPLC by New England Peptide LLC: p11C (CTPYDINQM), p54AS (TVPWPNASL), p54E660 (TVPWPNETL), p68A (STPPLVRLV), and TL8 (TTPESANL). The monomers and tetramers were prepared as previously described [85] (link), [86] (link). Tetramers were prepared using either streptavidin-PE (Prozyme), -APC (Prozyme), -AlexaFluor488 (Invitrogen), or -Qdot655 (Invitrogen). Monomers used in surface plasmon resonance studies were further quantified using an RC DC protein kit (Bio-Rad).

Immediately after the liver biopsy, cells were recovered by mechanical disruption, and immunostained for flow cytometric analysis. The leucocytes of blood sample were extracted with Filcool and the same protocol for immunostaining as for liver samples was used. Immuno-phenotyping was performed on fresh PBMC and liver samples with the following anti-human antibodies: CD3-PE-Cy7, CD56-PE from Beckman Coulter, CD56-FITC, IFN-γ-PE, Perforin-FITC and CD107a-APC from Becton-Dickinson Biosciences, CD3-APC-Cy7, CD45-APC-Cy7, uncoupled CD107a and CD107a Pacific Blue from Biolegend analyses were done as previously described [7] (link), [30] (link). Data were acquired on BD LSRII flow cytometer (BD Biosciences) and analyzed using FCS Express V3 software.

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: CD3- PE-Cy7 (Beckman Coulter, USA, clone UCHT1), CD56-APC (Beckman Coulter, USA, clone N901), CD56-Brilliant Violet 421 (Sony, USA, clone HCD56), CD56-PE (Beckman Coulter, USA, clone N901 (HLDA6)), CD57-PE (eBioscience, USA, clone TB01), CD57-FITC (Miltenyi Biotech, Germany, clone TB03), CD57-APC (Miltenyi Biotech, Germany, TB03), CD16-PE (Sony, USA) anti-NKG2A-PE (R&D Systems, USA, clone 131411), anti-KIR2DL2/DL3-PE (Miltenyi Biotech, Germany, clone DX27), anti-HLA-DR-FITC (Beckman Coulter, USA, clone B8.12.2). Cells were incubated in PBS containing 0.5% BSA (bovine serum albumin) and 0.01% sodium azide (labeling buffer) with mAbs for 30 min on ice, then washed twice with labeling buffer and centrifugation. Samples were subsequently analyzed using FACSCalibur flow cytometer (BD Biosciences, San Jose CA, USA) equipped with 488 and 640 nm lasers. At least 30000 events were recorded in lymphocyte gate for total NK cell population and 5000 events for NK cell clones. Acquired data was analyzed using Flowing Software version 2.5.1 (PerttuTerho, Turku Centre for Biotechnology, Finland) and FlowJo software version 7.6 (FlowJo LLC, Ashland, OR, USA). For fluorescence-activated cell sorting, cells were labeled with mAbs in PBS containing 0.5% BSA and 2 mM EDTA.