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Anti cd56 pecy7 b159

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Anti-CD56-PeCy7 (B159) is a monoclonal antibody that binds to the CD56 antigen. CD56 is expressed on natural killer (NK) cells, a subset of T cells, and some other cell types. The antibody is conjugated with the PeCy7 fluorescent dye, which can be used for flow cytometric analysis.

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2 protocols using anti cd56 pecy7 b159

1

NK Cell-Mediated Cytotoxicity Assay

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NK cells were isolated from blood by negative MACS selection (Miltenyi Biotec) according to manufacture's instructions. NK cells were activated with 1000 U/ml IL-2 (Proleukin) for 6 h at 21 % O2. Activated NK cells were co-cultured with target cells at 1:1 ratio in 96-well round-bottom plates in duplicate with anti-CD107a-Horizon-V450 (H4A3, BD). After 1 h, co-culture at 21 % O2 or 0.6 % O2, monensin (BD GolgiStop, Cat# 554724) was added. After another 8–9 h, co-cultures were stained on ice with anti-CD3-APC/H7 (SK7, BD), anti-CD56-PeCy7 (B159, BD), anti-KIR2DL1-APC (143211, R&D), anti-KIR2DL2/3/S2-PE (DX27, Miltenyi Biotec), anti-KIR3DL1-FITC (DX9, Miltenyi Biotec) and anti-NKG2A-PC5.5 (Z199, Beckman Coulter). For co-cultures with IL-2-activated NK cells, target cells were pre-incubated for 6 h at 21 % O2 or 0.6 % O2. For experiments without IL-2 activation, target cells cultured at 21 % O2 were used. Target cells were subsequently co-cultured with freshly isolated NK cells in a 10- to 12-h degranulation assay. For HLA-E blocking experiments, target cells were pre-incubated for 30 min at 37 °C with 10 µg/ml of anti-HLA-E (3D12; IgG1 isotype eBioscience) or IgG1 isotype control. Gating strategy is described in supplemental figure S2.
Sarkar S., van Gelder M., Noort W., Xu Y., Rouschop K.M., Groen R., Schouten H.C., Tilanus M.G., Germeraad W.T., Martens A.C., Bos G.M, & Wieten L. (2015). Optimal selection of natural killer cells to kill myeloma: the role of HLA-E and NKG2A. Cancer Immunology, Immunotherapy, 64(8), 951-963.
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2

Thyroid Immune Cell Profiling

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The samples were ex-vivo aspirated from the resected thyroid glands in the operation room and immediately mixed in RPMI media containing 10% FCS at 4°C. The red blood cells were lysed by a brief hypotonic shock. For all flow cytometry experiments, cells were stained with fluorochrome-conjugated antibodies against human CD3, CD4, CD8, CD14, CD19, CD25, CD45, CD56, and iNKT (anti-CD3-allophycocyanin/HIT3a, anti-CD4-APC-cy7/RPA-T4, anti-CD8-PE-cy5/HIT8a, anti-CD14-PE/M5E2, anti-CD19-PE-cy5/HIB19, anti-CD25-PE/M-A251, anti-CD45-FITC/HI30, anti-CD56-PE-cy7/B159, anti-iNKT FITC/6B11, TCRαβ-FITC/T10B9.1A-31 and TCRγδ-PE/B1; BD Biosciences) or isotype controls in serum-containing media. Freshly isolated single cells were incubated with antibodies for 20 minutes on ice for surface staining, washed and fixed in 1% paraformaldehyde. A subset of cells was permeabilized with cytofix/cytosperm fixation and permeabilization solution (BD Biosciences) and stained with fluorochrome-conjugated antibodies against human IL17, IFNg and Foxp3 (anti-IL17-PE/N49-653, anti-IFNg-PE/25723.11, anti- Foxp3-Alexa Fluor 488/259D/C7; BD Biosciences). Cells were also stained with Hoechst 33342 (10μg/ml for 2 hr, Hoechst fluorescence, 350 nm excitation/ 450 nm emission, linear scale) used to gate live cells containing 2n-4n cellular DNA.
Imam S., Paparodis R., Sharma D, & Jaume J.C. (2014). Lymphocytic Profiling in Thyroid Cancer Provides Clues for Failure of Tumor Immunity. Endocrine-related cancer, 21(3), 505-516.
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