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FITC Rat Anti-Human IL-4
FITC Rat Anti-Human IL-4
Expression of IL-4 by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with soluble anti-human CD3 antibody (1 µg/ml final concentration; Cat. No. 555329), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL-4 (10 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL-4 for 3 days. Finally, the cells were harvested and stimulated for 6 hours with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of GolgiStop™ (2µM final concentration; Cat. No. 554724). The cells were harvested, stained with PE-Cy5™-anti CD4 (.25 µg final concentration; Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of FITC-rat anti-human IL-4 antibody (FITC-MP4-25D2, Cat. No. 554484) by using the BD  Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding of FITC-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabelled MP4-25D2 antibody (2.5 µg, Cat. No. 554482; right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence controls and verified using unlabelled  antibody blocking controls.
Expression of IL-4 by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with soluble anti-human CD3 antibody (1 µg/ml final concentration; Cat. No. 555329), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL-4 (10 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL-4 for 3 days. Finally, the cells were harvested and stimulated for 6 hours with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of GolgiStop™ (2µM final concentration; Cat. No. 554724). The cells were harvested, stained with PE-Cy5™-anti CD4 (.25 µg final concentration; Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of FITC-rat anti-human IL-4 antibody (FITC-MP4-25D2, Cat. No. 554484) by using the BD  Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding of FITC-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabelled MP4-25D2 antibody (2.5 µg, Cat. No. 554482; right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence controls and verified using unlabelled  antibody blocking controls.
Product Details
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BD Pharmingen™
Human (QC Testing)
Rat IgG1
Purified Recombinant Human IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
0.5 mg/ml
AB_395423
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 FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometry Analysis: The FITC-conjugated MP4-25D2 antibody can be used for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-4-producing cells within mixed cell populations (see image, left panel). For optimal immunofluorescent staining for flow cytometric analysis, this antibody should be titrated (≤ 0.5 µg mAb/million cells). A useful control for demonstrating specificity of staining is the following: pre-block the fixed/permeabilized cells with unlabelled MP4-25D2 antibody (Cat. No. 554482) prior to staining. The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is FITC-R3-34 immunoglobulin (Cat. No. 554684); use at comparable concentrations to antibody of interest (e.g. , ≤ 0.5 µg mAb/ 1 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.

ELISA Detection: The biotinylated MP4-25D2 (Cat. No. 554483) antibody is useful as a detection antibody for a sandwich ELISA that measures human IL-4 protein levels. Biotinylated MP4-25D2 antibody can be paired with the purified 8D4-8 antibody (Cat. No. 554515) as the capture antibody, with recombinant human IL-4 protein (Cat. No. 554605) as the standard. For assay of IL-4 in serum or plasma, the BD OptEIA™ Human IL-4 ELISA Set (Cat. No. 555194) or  BD OptEIA™ Human IL-4 ELISA Kit (Cat. No. 550614) is recommended.

Neutralization: The NA/LE format of the MP4-25D2 antibody (Cat. No. 554481) has been reported to be useful for neutralization of human IL-4 bioactivity. A suitable NA/LE™ rat IgG1 isotype control to match the NA/LE™ MP4-25D2 antibody is the R3-34 antibody, Cat. No. 554683.

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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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 routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.

554484 Rev. 1
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
FITC
Blue 488 nm
494 nm
518 nm
554484 Rev.1
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: Neutralization). View Reference
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554484 Rev. 1

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