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      PE-CF594 Rat Anti-Human IL-4
      565161Image1.png

      Expression of IL-4 by stimulated human peripheral blood CD4+ T cells. An enriched preparation of peripheral blood CD4+ T 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, recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and human IL-4 (25 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and human IL-4 proteins for 3 days. Finally, the cells were harvested and stimulated for 5 h with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655).

             The fixed cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD4 (Cat. No. 561841/555349/561840) and either BD Horizon™ PE-CF594 Rat IgG1 κ Isotype Control (Cat. No. 562309; Left Panel) or BD Horizon PE-CF594 Mouse Anti-Human IL-4 antibody (Cat. No. 565161; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric contour plots showing the correlated expression of IL-4 (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

      hIL4_PECF594_vs_CD4.png

      Expression of IL-4 by stimulated human peripheral blood CD4+ T cells. An enriched preparation of peripheral blood CD4+ T 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, recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and human IL-4 (25 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and human IL-4 proteins for 3 days. Finally, the cells were harvested and stimulated for 5 h with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655).

             The fixed cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD4 (Cat. No. 561841/555349/561840) and either BD Horizon™ PE-CF594 Rat IgG1 κ Isotype Control (Cat. No. 562309; Left Panel) or BD Horizon PE-CF594 Mouse Anti-Human IL-4 antibody (Cat. No. 565161; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric contour plots showing the correlated expression of IL-4 (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

      565161Image1.png hIL4_PECF594_vs_CD4.png Orange-Cap-Blue-Label.jpg

      Expression of IL-4 by stimulated human peripheral blood CD4+ T cells. An enriched preparation of peripheral blood CD4+ T 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, recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and human IL-4 (25 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and human IL-4 proteins for 3 days. Finally, the cells were harvested and stimulated for 5 h with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655).

             The fixed cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD4 (Cat. No. 561841/555349/561840) and either BD Horizon™ PE-CF594 Rat IgG1 κ Isotype Control (Cat. No. 562309; Left Panel) or BD Horizon PE-CF594 Mouse Anti-Human IL-4 antibody (Cat. No. 565161; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric contour plots showing the correlated expression of IL-4 (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

      Expression of IL-4 by stimulated human peripheral blood CD4+ T cells. An enriched preparation of peripheral blood CD4+ T 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, recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and human IL-4 (25 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and human IL-4 proteins for 3 days. Finally, the cells were harvested and stimulated for 5 h with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655).

             The fixed cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD4 (Cat. No. 561841/555349/561840) and either BD Horizon™ PE-CF594 Rat IgG1 κ Isotype Control (Cat. No. 562309; Left Panel) or BD Horizon PE-CF594 Mouse Anti-Human IL-4 antibody (Cat. No. 565161; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric contour plots showing the correlated expression of IL-4 (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

      Product Details
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      BD Horizon™
      Interleukin-4; IL4; BCGF-1; BSF-1; Lymphocyte stimulatory factor 1
      Human (QC Testing)
      Rat IgG1
      Purified Recombinant Human IL-4
      Intracellular staining (flow cytometry) (Routinely Tested)
      5 µl
      AB_2739086
      Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with BD Horizon™ PE-CF594 under optimum conditions, and unconjugated antibody and free PE-CF594 were removed.
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      Product Notices

      1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
      2. An isotype control should be used at the same concentration as the antibody of interest.
      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.
      4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      5. When excited by the yellow-green (561-nm) laser, the fluorescence may be brighter than when excited by the blue (488-nm) laser.
      6. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using multi-laser cytometers, which may directly excite both PE and CF™594.
      7. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
      8. CF™ is a trademark of Biotium, Inc.
      9. This product is provided under an Agreement between BIOTIUM and BD Biosciences. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications owned or licensed by Biotium, Inc. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
      10. Texas Red is a registered trademark of Molecular Probes, Inc., Eugene, OR.
      11. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      12. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      565161 Rev. 1
      Antibody Details
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      MP4-25D2

      The MP4-25D2 monoclonal antibody specifically binds to Interleukin-4 (IL-4). IL-4 is also known as Lymphocyte stimulatory factor 1, B cell stimulatory factor 1 (BSF-1), or B cell growth factor 1 (BCGF-1). IL-4 is produced by activated T cells, basophils, and mast cells. IL-4 is a multifunctional cytokine and growth factor that affects a variety of different target cell types. IL-4 can costimulate T cell proliferation and survival, as well as help drive Th2-like cell differentiation and effector functions. It costimulates B cell proliferation and survival, and can help B cells differentiate into IgG4- or IgE-producing cells. IL-4 can also diminish the proinflammatory functions of monocytes and macrophages. IL-4 exerts its biological effects by binding with high affinity to cell surface CD124 (IL-4Rα chain). CD124 forms signaling IL-4 receptor complexes with either CD132/common γ chain or CD213a1/IL-13Rα1 to form either Type I or Type II IL-4 Receptor complexes, respectively. The immunogen used to generate the MP4-25D2 hybridoma was purified recombinant human IL-4. This is a neutralizing antibody. The MP4-25D2 antibody has been reported to crossreact with IL-4 from rhesus monkeys. The use of the MP4-25D2 antibody for epitope mapping of human IL-4 has been described.

      This antibody is conjugated to BD Horizon PE-CF594, which has been developed exclusively by BD Biosciences as a better alternative to PE-Texas Red®. PE-CF594 excites and emits at similar wavelengths to PE-Texas Red® yet exhibits improved brightness and spectral characteristics. Due to PE having maximal absorption peaks at 496 nm and 564 nm, PE-CF594 can be excited by the blue (488-nm), green (532-nm) and yellow-green (561-nm) lasers and can be detected with the same filter set as PE-Texas Red® (eg 610/20-nm filter).

      565161 Rev. 1
      Format Details
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      PE-CF594
      BD Horizon™ PE-CF594 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye with an emission maximum (Em Max) at 615-nm. PE-CF594, driven by BD innovation, 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 615 nm (e.g., a 610/20-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the green (532-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      altImg
      PE-CF594
      496 nm, 566 nm
      615 nm
      565161 Rev.1
      Citations & References
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      View product citations for antibody "565161" on CiteAb

      Development References (5)

      1. 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, Neutralization). View Reference
      2. Chretien I, Van Kimmenade A, Pearce MK, Banchereau J, Abrams JS. Development of polyclonal and monoclonal antibodies for immunoassay and neutralization of human interleukin-4. J Immunol Methods. 1989; 117(1):67-71. (Clone-specific: ELISA, Neutralization). View Reference
      3. Jung T, Schauer U, Rieger C, et al. Interleukin-4 and interleukin-5 are rarely co-expressed by human T cells. Eur J Immunol. 1995; 25(8):2413-2416. (Clone-specific: Flow cytometry). View Reference
      4. 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
      5. Ramanathan L, Ingram R, Sullivan L, et al. Immunochemical mapping of domains in human interleukin 4 recognized by neutralizing monoclonal antibodies. Biochemistry. 1993; 32(14):3549-3556. (Clone-specific: Neutralization). View Reference
      View All (5) View Less
      565161 Rev. 1

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      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.