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Two-color flow cytometric analysis of IL-10 expression in stimulated Human lymphocytes. Human peripheral blood mononuclear cells were stimulated with immobilized Purified NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 µg/ml for plate coating) and soluble Purified NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 2 µg/ml) antibodies and Recombinant Human IL-2 (Cat. No. 554603; 10 ng/ml) and Human IL-4 (Cat. No. 554605; 25 ng/ml) proteins for 2 days. The stimulated cells were washed and cultured in medium containing IL-2 and IL-4 for another 3 days. Finally, the cells were harvested and cultured (5 hr) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) either without (Unstimulated; Left Plot) or with (Restimulated; Right Plot) Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed, permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with BD Horizon™ RY586 Mouse Anti-Human IL-10 and Viral IL-10 antibody (Cat. No. 568520/568521; Right Plot) using BD Biosciences Intracellular Cytokine Staining protocol. Pseudocolor density plots showing the correlated expression of IL-10 versus side light-scatter signals (SSC-A) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 Cell Analyzer System and FlowJo™ software.
BD Horizon™ RY586 Rat Anti-Human and Viral IL-10
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The JES3-9D7 monoclonal antibody specifically reacts with human IL-10 (Interleukin-10) and viral IL-10. The immunogen used to generate the JES3-9D7 hybridoma was recombinant human IL-10 expressed by COS cells. IL-10 is also known as CSIF (Cytokine synthesis inhibitory factor) and (TGIF) T-cell growth inhibitory factor. IL-10 is expressed by various cell types including activated monocytes, macrophages, dendritic cells, mast cells, granulocytes, and lymphocytes. IL-10 is a pleiotropic cytokine that can downregulate proinflammatory immune responses, such as Th1-like responses, while promoting other responses including B cell proliferation and antibody production.
Development References (7)
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Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
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Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific: ELISA). View Reference
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Burdin N, Peronne C, Banchereau J, Rousset F. Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10. J Exp Med. 1993; 177(2):295-304. (Clone-specific: ELISA). View Reference
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Gotlieb WH, Abrams JS, Watson JM, Velu TJ, Berek JS, Martinez-Maza O. Presence of interleukin 10 (IL-10) in the ascites of patients with ovarian and other intra-abdominal cancers. Cytokine. 1992; 4(5):385-390. (Clone-specific: ELISA). View Reference
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Jeffery LE, Burke F, Mura M, et al. 1,25-Dihydroxyvitamin D3 and IL-2 combine to inhibit T cell production of inflammatory cytokines and promote development of regulatory T cells expressing CTLA-4 and FoxP3.. J Immunol. 2009; 183(9):5458-67. (Clone-specific: Flow cytometry). View Reference
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Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Methodology: IC/FCM Block). View Reference
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Yssel H, De Waal Malefyt R, Roncarolo MG, et al. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J Immunol. 1992; 149(7):2378-2384. (Clone-specific: ELISA). View Reference
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