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BUV737 Rat Anti-Human IL-4
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This product is the replacement for [564689].
BUV737 Rat Anti-Human IL-4

Expression of IL-4 by stimulated human peripheral blood mononuclear cells. 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, recombinant human IL-2 (10 ng/ml; Cat. No. 554603) and human IL-4 (25 ng/ml; Cat. No. 554605) proteins for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and IL-4 for 3 days. Finally, the cells were harvested and stimulated for 5 hr with Phorbol 12-Myristate 13-Acetate (PMA; 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 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 PE Mouse Anti-Human CD4 antibody (Cat. No. 555347/561843/561844) and either BD Horizon™ BUV737 Rat IgG1 κ Isotype Control (Cat. No. 612770; Left Plot) or BD Horizon BUV737 Mouse Anti-Human IL-4 antibody (Cat. No. 612835; Right Plot) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot 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 cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.

Expression of IL-4 by stimulated human peripheral blood mononuclear cells. 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, recombinant human IL-2 (10 ng/ml; Cat. No. 554603) and human IL-4 (25 ng/ml; Cat. No. 554605) proteins for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and IL-4 for 3 days. Finally, the cells were harvested and stimulated for 5 hr with Phorbol 12-Myristate 13-Acetate (PMA; 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 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 PE Mouse Anti-Human CD4 antibody (Cat. No. 555347/561843/561844) and either BD Horizon™ BUV737 Rat IgG1 κ Isotype Control (Cat. No. 612770; Left Plot) or BD Horizon BUV737 Mouse Anti-Human IL-4 antibody (Cat. No. 612835; Right Plot) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot 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 cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.

Product Details
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BD Horizon™
Interleukin-4; IL4; BCGF-1; BSF-1; Lymphocyte stimulatory factor 1
Human (QC Testing), Rhesus (Reported)
Rat IgG1
Purified Recombinant Human IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
Aqueous buffered solution containing ≤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 BUV737 under optimum conditions, and unconjugated antibody and free BD Horizon BUV737 were removed.

Recommended Assay Procedures

        BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation).  When fluorochrome conjugated antibodies are bound to BD CompBeads, they have spectral properties very similar to cells.   However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls.  It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.
        For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment.  Fluorescent dye interactions may cause staining artifacts which may affect data interpretation.  The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions.  More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
        Note:  When using high concentrations of antibody, background binding of this dye to erythroid cell subsets (mature erythrocytes and precursors) has been observed.  For researchers studying these cell populations, or in cases where light scatter gating does not adequately exclude these cells from the analysis, this background may be an important factor to consider when selecting reagents for panel(s).

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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. BD Horizon Brilliant Ultraviolet 737 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,575,303; 8,354,239.
  6. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
612835 Rev. 2
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. 

The antibody was conjugated to BD Horizon BUV737 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome with an Ex Max near 350 nm and an Em Max near 737 nm. BD Horizon Brilliant BUV737 can be excited by the ultraviolet laser (355 nm) and detected with a 740/35 nm filter. Due to the excitation of the acceptor dye by the red laser line, there may be significant spillover into red laser detectors with filters in the 700-720 nm range.

612835 Rev. 2
Format Details
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BUV737
The BD Horizon Brilliant™ Ultraviolet 737 (BUV737) Dye is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This tandem fluorochrome is comprised of a BUV395 donor with an excitation maximum (Ex Max) of 350-nm and an acceptor dye with an emission maximum (Em Max) at 735-nm. BUV737, driven by BD innovation, is designed to be excited by the ultraviolet laser (355-nm) and detected using an optical filter centered near 740-nm (e.g., 740/35 bandpass filter). The acceptor dye can be excited by the Red (628–640nm) 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.
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BUV737
Ultraviolet 355 nm
350 nm
735 nm
612835 Rev.2
Citations & References
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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: Functional assay, Neutralization). View Reference
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612835 Rev. 2

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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.