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Anti il17a alexa647

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Anti-IL17A–Alexa647 is a fluorescently labeled antibody that binds to the cytokine interleukin-17A (IL-17A). It is designed for use in various immunological and cell biology applications that require the detection or quantification of IL-17A.

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4 protocols using anti il17a alexa647

1

Multiparametric Flow Cytometry Analysis

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Cells were washed in PBS + 10% FCS and stained with primary antibodies (anti-CD45RA-BV421, anti-CD62L-APC-Cy7, anti CDCD49f-FITC, anti-CD51-APC, anti CD493-FITC, anti-CD49b-APC, anti-CD49a-APC, all Biolegend; anti-IL17a-Alexa647 and anti-CD146/MCAM-PE, both BD Bioscience) for 30 min on ice. Intracellular staining of IL-17a was performed by implementation of the CytoFAST Fix/Perm buffer set (Biolegend) exactly according to the manufacturer’s instructions. The cells were fixed in 4% PFA (Sigma) and the stainings were assessed on a BD Canto II flow cytometer.
Zondler L., Herich S., Kotte P., Körner K., Schneider-Hohendorf T., Wiendl H., Schwab N, & Zarbock A. (2020). MCAM/CD146 Signaling via PLCγ1 Leads to Activation of β1-Integrins in Memory T-Cells Resulting in Increased Brain Infiltration. Frontiers in Immunology, 11, 599936.
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2

Intracellular Cytokine Profiling of T Cells

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For intracellular cytokine staining for PBMC or CD4+ T cells, assays were carried out with staining buffers and antibodies from BD Biosciences. Briefly, cells were seeded into a 96-well plate (up to 1 × 106 cell per well) and stimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) in the presence of GolgiStop for 4 h. After stimulation, cells were fixed with BD Cytofix fixation buffer and washed with BD Perm/Wash buffer. Cells in each well were equally divided into two wells, with one for intracellular cytokine staining and the other for isotype control staining. The following fluorophore-conjugated antibodies from BD Biosciences were used for staining analysis or as isotype controls: anti-CD4-pacific blue (clone: RPA-T4; 1:330), anti-IL-17A-Alexa647 (clone: N49-653; 1:20), anti-IFN-γ-FITC (clone: B27: 1:100), anti-IL-10-PE (clone: JES3-19F: 1:660), mouse IgG1-Alexa647 (clone: MOPC-21; 1:40), mouse IgG1-FITC (clone: MOPC-21; 1:100), and rat IgG2a-PE (clone: R35-95: 1:660). Stained cells were analyzed with a BD LSR II cytometer. Cytokine secretion in CD4+ lymphocytes was accessed with FlowJo.
Hu D., Notarbartolo S., Croonenborghs T., Patel B., Cialic R., Yang T.H., Aschenbrenner D., Andersson K.M., Gattorno M., Pham M., Kivisakk P., Pierre I.V., Lee Y., Kiani K., Bokarewa M., Tjon E., Pochet N., Sallusto F., Kuchroo V.K, & Weiner H.L. (2017). Transcriptional signature of human pro-inflammatory TH17 cells identifies reduced IL10 gene expression in multiple sclerosis. Nature Communications, 8, 1600.
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3

Cell Viability and Immune Cell Profiling

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Cell survival of MEFs was quantitated using the CellTiter-Glo Luminescent Cell Viability Assay per manufacturer’s instructions (Promega). The following antibodies were purchased (BD bioscience) and used for FACS studies: anti-CD4-PEcy7, anti-CD4-APC, anti-CD8-PEcy7, anti-CD8-APC, anti-CD11b–PEcy7, anti-Gr1-APC, anti-CD45.1-APC, anti-CD45.1-FITC, anti-CD45.2-APC, anti-CD45.2-FITC, anti-CD44-FITC, anti-CD62L–PE, anti-CD62L–APC, anti-TCRβ-FITC. Live cells were quantitated by flow cytometry of DAPI-negative cells. For intracellular cytokine expression, cells were incubated with PMA (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich) and GolgiPlug (1 µl/ml, BD Bioscience) for 4 hrs before harvesting. Cells were stained using anti-CD4-e450 (Tonbo), anti-IFNγ−PE (BD Bioscience), anti-IL17A–Alexa647 (BD Bioscience), Live/Dead cell stain kit (Invitrogen) and Cytofix/Cytoperm kit (BD Bioscience).
Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.
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4

Cell Viability and Immune Cell Profiling

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Cell survival of MEFs was quantitated using the CellTiter-Glo Luminescent Cell Viability Assay per manufacturer’s instructions (Promega). The following antibodies were purchased (BD bioscience) and used for FACS studies: anti-CD4-PEcy7, anti-CD4-APC, anti-CD8-PEcy7, anti-CD8-APC, anti-CD11b–PEcy7, anti-Gr1-APC, anti-CD45.1-APC, anti-CD45.1-FITC, anti-CD45.2-APC, anti-CD45.2-FITC, anti-CD44-FITC, anti-CD62L–PE, anti-CD62L–APC, anti-TCRβ-FITC. Live cells were quantitated by flow cytometry of DAPI-negative cells. For intracellular cytokine expression, cells were incubated with PMA (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich) and GolgiPlug (1 µl/ml, BD Bioscience) for 4 hrs before harvesting. Cells were stained using anti-CD4-e450 (Tonbo), anti-IFNγ−PE (BD Bioscience), anti-IL17A–Alexa647 (BD Bioscience), Live/Dead cell stain kit (Invitrogen) and Cytofix/Cytoperm kit (BD Bioscience).
Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.
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