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Expression of IL-2 by stimulated CD4+ and CD4- BALB/c spleen cells. Splenocytes from 6 month old BALB/c mice were stimulated for 5 hours with hamster anti-mouse CD3 (2 µg/ml final concentration; clone 145-2C11, Cat. No. 553057) and hamster anti-mouse CD28 (2 µg/ml final concentration; clone 37.51, Cat. No. 553294) antibodies in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The splenocytes were harvested, stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.25 µg of PE anti-mouse IL-2 (Cat. No. 554428) (left panel). To demonstrate specificity, PE-JES6-5H4 staining was blocked by each of the following: 1) preincubation of the conjugated antibody with recombinant mouse IL-2 (0.25 µg, Cat. No. 550069; middle panel) and by 2) preincubation of the fixed/permeabilized cells with unlabelled JES6-5H4 mAb (5.0 µg; Cat. No. 554425) prior to staining with PE-JES6-5H4 (right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
BD Pharmingen™ PE Rat Anti-Mouse IL-2
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
Recommended Assay Procedure:
Immunofluorescent Staining for Flow Cytometric Analysis: The PE conjugated JES6-5H4 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-2 producing cells within mixed cell populations. For optimal immunofluorescent staining for flow cytometric analysis this antibody should be titrated. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or to the chapter on intracellular staining in the Immune Function Handbook. A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the PE-conjugated JES6-5H4 antibody with ligand (e.g., recombinant mouse IL-2, Cat No. 550069) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled JES6-5H4 antibody (Cat. No. 554425) prior to staining. The staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe.
A suitable rat IgG2b isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse cells is PE-A95-1 (Cat. No. 556925); use at comparable concentrations to antibody of interest.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Use of these products to measure activation antigens expressed on mononuclear cell subsets for the purpose of monitoring immunoregulatory status can fall under one or more claims of the following patents: US Patent Nos. 5,445,939, 5,656,446, 5,843,689; European Patent No. 319,543; Canadian Patent No. 1,296,622; Australian Patent No. 615,880; and Japanese Patent No. 2,769,156.
Companion Products
The JES6-5H4 monoclonal antibody specifically binds to mouse interleukin-2 (IL-2), a multifunctional cytokine that plays pivotal roles in immunity and tolerance. It is produced by activated T cells and affects the activation, growth, proliferation and/or differentiation of various cell types including T and B lymphocytes and their precursors, LAK cells, NK cells, and monocytes/macrophages. IL-2 mediates its biological activities by binding to IL-2 receptor complexes. The intermediate affinity IL-2R is comprised of IL-2Rβ (CD122) and common gamma chain (γc; CD132) subunits, whereas the high-affinity IL-2R is comprised of IL-2Rα (CD25), IL-2Rβ, and γc subunits. The JES6-5H4 monoclonal antibody binds to IL-2 and neutralizes its biological activity.
Development References (4)
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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). 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, Immunoprecipitation). 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: Flow cytometry). View Reference
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Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Clone-specific: ELISA). View Reference
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.