Cd161 pe

Manufactured by Miltenyi Biotec

CD161-PE is a fluorochrome-conjugated monoclonal antibody that binds to the CD161 antigen. CD161 is a type II transmembrane glycoprotein receptor expressed on the surface of natural killer (NK) cells, a subset of T cells, and other immune cell types. The PE (phycoerythrin) fluorochrome allows for the detection and analysis of CD161-positive cells using flow cytometry and other immunoassay techniques.

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Close spelling variants of this product

Anti-CD161-PE-Vio770 CD161-PE and CD161-APC

7 protocols using cd161 pe

Comprehensive Immune Cell Profiling by Flow Cytometry

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Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation with Lymphoprep (Axis-Shield). PBMC or whole blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-efluor605, CD4-efluor450, CD27-APC-efluor780, HLA-DR-efluor780, CD45RA-efluor605, FOXP3-PE (eBioscience), CD4-APC-H7, CD8-Percp, CD8-PE-Cy7, CD31-AF647, CD45RO-FITC, CD45RO-PE-Cy7, CCR7-PE-Cy7, Ki-67-Percp-cy5.5, CTLA-4-BV421 (BD Biosciences), PD-1-PE, CD28-AF700 (Biolegend), and CD161-PE (Miltenyi Biotec). Intracellular staining for FOXP3, Helios, Ki-67, and CTLA-4 was performed after cells were permeabilized with a FOXP3 staining buffer set according to instructions of the manufacturer (eBioscience). Whole blood samples were treated with BD lysing solution according to the instructions of the manufacturer (BD Biosciences). Stained samples were analyzed on a LSR-II flow cytometer (BD Biosciences). Analysis was performed with Kaluza Flow Analysis Software (Beckman Coulter). Absolute numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells were determined according to the MultiTest TruCount method (BD Biosciences), as described by the manufacturer. TruCount samples were measured on a FACSCanto-II (BD Biosciences) and analyzed with FACSCanto Clinical Software (BD Biosciences).

van den Brom R.R., van der Geest K.S., Brouwer E., Hospers G.A, & Boots A.M. (2018). Enhanced expression of PD-1 and other activation markers by CD4+ T cells of young but not old patients with metastatic melanoma. Cancer Immunology, Immunotherapy, 67(6), 925-933.

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Tonsillar Cell Stimulation and Evaluation

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Tonsillar cells were stained with CD161-PE (Milteny Biotec) and depleted from the sample by FACS-sroting, using MoFlo™ (Beckman Coulter). After CpG (0.5μM, Invivogen) or anti-CD40 (5μg/ml, Macbeth) + IL-4 stimulation (50ng/ml, PreproTech), levels of CD38 and CD83, respectively, were measured in alive CD19+ cells.

Llibre A., López-Macías C., Marafioti T., Mehta H., Partridge A., Kanzig C., Rivellese F., Galson J.D., Walker L.J., Milne P., Phillips R.E., Kelly D.F., Freeman G.J., El Shikh M.E., Klenerman P, & Willberg C.B. (2016). LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation. The Journal of Immunology Author Choice, 196(5), 2085-2094.

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Multiparametric Flow Cytometry Analysis

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Blood samples (EDTA) were stained with the following monoclonal antibodies: CD3-eFluor605, CD4-eFluor450, TCRγδ-PE (eBioscience), CD45RO-FITC, CCR7-PE-Cy7, CD4-PerCP, CD8-PerCP, CD8-APC-H7, DNAX accessory molecule-1 (DNAM-1)-FITC (BD), CD161-PE, CD161-APC (Miltenyi), 2B4-PE, NKG2D-PE-Cy7, TCR-Vα7.2-FITC, TCR-Vα24-Jα18-FITC (Biolegend), TCR-Vβ11-PE (Beckman Coulter), and KLRG1-FITC (generous gift from H. Pirchner). An overview of antibody panels is shown in Table S1 in Supplementary Material. Samples were subsequently treated with BD Lysing Solution (BD Biosciences) according to instructions of the manufacturer. Samples were measured on a LSR-II flow cytometer (BD) and analyzed with Kaluza Analysis Software (Beckman Coulter). In addition, the absolute numbers of circulating lymphocyte subsets were determined according to the MultiTest TruCount method (BD), as described by the manufacturer. Data were acquired on a FACSCanto-II flow cytometer (BD) and analyzed with FACSCanto Clinical Software (BD). The number of events for a particular T cell population needed to be more than 100 to allow for subsequent analysis of cellular markers, cytokines, and cytotoxic molecules.

van der Geest K.S., Kroesen B.J., Horst G., Abdulahad W.H., Brouwer E, & Boots A.M. (2018). Impact of Aging on the Frequency, Phenotype, and Function of CD161-Expressing T Cells. Frontiers in Immunology, 9, 752.

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Comprehensive Immune Cell Profiling

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Antibodies/dyes and dilutions used were: viability dye live/dead fixable-violet (L34955, Invitrogen, 1:1250), CD3-eFluor450 (48-0038, eBioscience, 1:100), CD3-PECy7 (25-0038, eBioscience, 1:100), CD4-VioGreen (130-096-900, Miltenyi Biotech, 1:50), CD8-VioGreen (130-098-062, Miltenyi Biotech, 1:50), CD8-V450 (560347, BD, 1:50), CD8-PerCP.Cy5.5/PerCP (301032, Biolegend, 1:100), CD38-APC (555462, BD, 1:50), CD69-FITC (11-0699, eBioscience, 1:40), CD161-APC (130-098-908, Miltenyi Biotech, 1:100), CD161-PE (130-099-193, Miltenyi Biotech, 1:100), IFN-γ-FITC (130-091-641, Miltenyi Biotech, 1:50), IFN-γ-APCCy7 (502529 Biolegend, 1:50), Vα7.2-PE/PeCy7/APC/FITC (351705/351711/351707/351703, Biolegend, 1:50). Granzyme B-APC (MHGB05, Invitrogen), IL-18Ra-APC (17-7183-41, eBioscience, 1:50), TNF-α-PeCy7 (502929, Biolegend, 1:100). All data was acquired on a MACSQuant (Miltenyi Biotech) or a BD FACSVerse (BD) and analyzed on FlowJo (Tree Star Inc.). Gating strategy is shown in Supplementary Fig. 12.

van Wilgenburg B., Scherwitzl I., Hutchinson E.C., Leng T., Kurioka A., Kulicke C., de Lara C., Cole S., Vasanawathana S., Limpitikul W., Malasit P., Young D., Denney L., Moore M.D., Fabris P., Giordani M.T., Oo Y.H., Laidlaw S.M., Dustin L.B., Ho L.P., Thompson F.M., Ramamurthy N., Mongkolsapaya J., Willberg C.B., Screaton G.R, & Klenerman P. (2016). MAIT cells are activated during human viral infections. Nature Communications, 7, 11653.

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Tonsil B Cell Activation Assay

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Tonsillar cells were stained with CD161-PE (Miltenyi Biotec) and depleted from the sample by FACS sorting using a MoFlo (Beckman Coulter). After CpG (0.5 μM, InvivoGen) or anti-CD40 (5μg/ml, Macbeth) plus IL-4 stimulation (50 ng/ml, PeproTech), levels of CD38 and CD83, respectively, were measured in alive CD19⁺ cells.

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Immunophenotyping of IL-17-Associated Diseases

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Peripheral blood of patients affected by IL-17 correlated disease was drawn after their informed consent (see above). PBMCs were obtained after gradient stratification on Ficoll-Paque Plus (GE Healthcare); cells were stained with monoclonal antibodies anti CD3 Percp, CD4 Fitc, CD161 Pe (Miltenyi biotech) for FACS analysis; PBMCs mRNA was also extracted and reverse transcribed for qPCR Real Time analysis, as described below. Sera were obtained according to a standard protocol, by blood centrifugation, and S1P levels were measured as below described.

Barra G., Lepore A., Gagliardi M., Somma D., Matarazzo M.R., Costabile F., Pasquale G., Mazzoni A., Gallo C., Nuzzo G., Annunziato F., Fontana A., Leonardi A, & De Palma R. (2018). Sphingosine Kinases promote IL-17 expression in human T lymphocytes. Scientific Reports, 8, 13233.

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Multicolor Flow Cytometry Immunophenotyping

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For external staining, cells from cell lines or PBMCs in PBS were incubated with anti-surface antibodies at room temperature (RT) for 20 min. Live/dead staining was performed using LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit(Invitrogen), at 633 or 635 nm excitation.
For internal staining, cells were fixed with 2% formaldehyde (Sigma Aldrich) in PBS for 10 min and permeabilized with IX permeabilization buffer (eBioscience) in water.
The following antibodies were used: CD3-FITC (BioLegend, Catalog No. 300406, clone UCHT1, Mouse IgG1, k), CD8-PerCP-Cy5.5 (BioLegend, Catalog No. 344710, clone SK1, Mouse IgG1, k), CD38-PerCP-Cy5.5 (BioLegend, Catalog No. 303522, clone HIT2, Mouse IgG1, k), CD56-APC (Biolegend, Catalog No. 318310, clone HCD56, Mouse IgG1, k); CD19-BV421 (BD Bioscience, Catalog No. 562441, clone HIB19, mouse IgG1, k); CD4-VioGreen (Miltenyi Biotec, Catalog No. 130-106-712, clone M-T466, Mouse IgG1, k), CD161-PE (Miltenyi Biotec, Catalog No. 130-092-677, clone 191B8, Mouse IgG2a), IgG2A isotype control (R&D Systems, Catalog No. MAB003, mouse); and 2H7 mAb. When non-conjugated primary antibodies were used, a secondary rat anti-mouse IgG2A-PE (R&D Systems, Catalog No. F0129, clone 344701, IgG1) was used.
FACS analysis was performed on Miltenyi Biotec MACSQuant cytometer and analyzed with FlowJo Version 9.6.2 software (TreeStar).

Llibre A., Garner L., Partridge A., Freeman G.J., Klenerman P, & Willberg C.B. (2016). Expression of lectin-like transcript-1 in human tissues. F1000Research, 5, 2929.

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