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Expression of IL-4 by stimulated CD4+ rat splenocytes. Purified splenic CD4+ cells from a 6 month old BN rat were stimulated with plate-bound anti-CD3 (G4.18, Cat. No. 554830 at 25 µg/ml) and soluble anti-rat CD28 (clone JJ319, Cat. No. 554993 at 2 µg/ml) for 2 days in culture together with rat IL-2 (10 ng/ml, Cat. No. 555106) and rat IL-4 (892 ng/ml, Cat. No. 555107), followed by a 3 day incubation with only rat IL-2 and rat IL-4. This was followed by a 6 h stimulation with PMA (1 ng/ml; Sigma, Cat. #P-8139) and Ionomycin (250 ng/ml; Sigma, Cat. #I-0634) in the presence of GolgiStop™ (Cat. No. 554724; aka monensin 2 µM). The cells were then fixed, permeabilized, and subsequently stained with 0.25 µg of PE-mouse anti-rat IL-4 antibody (Cat. No. 555082; far left panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of the PE-OX-81 antibody was blocked by preincubation of the PE-conjugated antibody with recombinant rat IL-4 (0.25 µg, Cat. No. 555107; center left panel), and by preincubation of the fixed/permeabilized cells with the unlabeled OX-81 antibody (5.0 µg, Cat. No. 555080; center right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control (far right panel), and verified with the recombinant cytokine blocking (center left panel) and unlabeled antibody blocking (center right panel) specificity controls.
NA PE Mouse Anti-Rat IL-4
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
Immunofluorescent Staining and Flow Cytometry: The PE-conjugated OX-81 antibody can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate IL-4 producing cells within mixed cell populations. For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/million cells). For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or 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 fluorochrome-conjugated OX-81 antibody with ligand (e.g., recombinant rat IL-4, Cat No. 555107) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabelled OX-81 antibody (Cat. No. 555080) prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized rat or human cells is PE MOPC-21 immunoglobulin (Cat. No. 554680); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/million cells).
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.
Companion Products
The OX-81 antibody reacts with rat interleukin-4 (IL-4). The immunogen used to generate this hybridoma was recombinant rat IL-4 protein. This is a neutralizing antibody.
This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
Development References (4)
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Prigent P, Saoudi A, Pannetier C, et al. Mercuric chloride, a chemical responsible for T helper cell (Th)2-mediated autoimmunity in brown Norway rats, directly triggers T cells to produce interleukin-4. J Clin Invest. 1995; 96(3):1484-1489. (Clone-specific: Neutralization). 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, IC/FCM Block). View Reference
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Ramirez F, Fowell DJ, Puklavec M, Simmonds S, Mason D. Glucocorticoids promote a TH2 cytokine response by CD4+ T cells in vitro. J Immunol. 1996; 156(7):2406-2412. (Immunogen: Neutralization). View Reference
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Saoudi A, Simmonds S, Huitinga I, Mason D. Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific T cells. J Exp Med. 1995; 182(2):335-344. (Clone-specific: Neutralization). 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.