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      PE Mouse Anti-Rat IL-4
      555082Image1.png

      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.

      555082Image1.png 24055A_555082_image4.png Red-Cap-Blue-Label.jpg

      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.

      Product Details
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      BD Pharmingen™
      Rat (QC Testing)
      Mouse IgG1, κ
      Recombinant rat IL-4 protein
      Intracellular staining (flow cytometry) (Routinely Tested)
      0.2 mg/ml
      AB_395688
      Aqueous buffered solution containing ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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      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).

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      Product Notices

      1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      3. 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.
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      555082 Rev. 2
      Antibody Details
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      OX-81

      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.

      555082 Rev. 2
      Format Details
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      PE
      R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      altImg
      PE
      496 nm, 566 nm
      576 nm
      555082 Rev.2
      Citations & References
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      View product citations for antibody "555082" on CiteAb

      Development References (4)

      1. 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
      2. 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
      3. 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
      4. 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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      555082 Rev. 2

      Please refer to Support Documents for Quality Certificates

       

      Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

       

      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.