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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; Cat. No. 555329), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and IL-4 (20 ng/ml; 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 h with PMA (Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 -anti CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.05 µg of PE-rat anti-human IL-4 antibody (PE-MP4-25D2, Cat. No. 554485) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled 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 unlabeled 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; Cat. No. 555329), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and IL-4 (20 ng/ml; 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 h with PMA (Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 -anti CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.05 µg of PE-rat anti-human IL-4 antibody (PE-MP4-25D2, Cat. No. 554485) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled 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 unlabeled 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; Cat. No. 555329), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and IL-4 (20 ng/ml; 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 h with PMA (Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 -anti CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.05 µg of PE-rat anti-human IL-4 antibody (PE-MP4-25D2, Cat. No. 554485) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled 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 unlabeled 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; Cat. No. 555329), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and IL-4 (20 ng/ml; 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 h with PMA (Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 -anti CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.05 µg of PE-rat anti-human IL-4 antibody (PE-MP4-25D2, Cat. No. 554485) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled 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 unlabeled antibody blocking controls.
Recommended Assay Procedure:
1. Blocking Control for Intracellular Staining: The unlabeled MP4-25D2 antibody can be used as a blocking control to demonstrate specificity of IL-4 staining by conjugated MP4-25D2 antibody (Cat. No. 554486, 554485, No. 554484). To perform this control, the fixed/permeabilized cells (~ 1 million) can be incubated with 1-10 µg of unlabeled MP4-25D2 antibody (Cat. No. 554482) for 20 minutes at 4°C, prior to staining with conjugated MP4-25D2 antibody. The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.
2. Neutralization: The NA/LE™ MP4-25D2 antibody (Cat. No. 554481) is useful for neutralization of human IL-4 bioactivity. A suitable NA/LE™ rat IgG1 isotype control to match the MP4-25D2 antibody is the R3-34 antibody, Cat. No. 554682.
3. WB: The purified MP4-25D2 antibody has been reported to be useful for Western blotting. Please note that this application is not routinely tested at BD Biosciences Pharmingen.
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 as a blocking control for intracellular staining. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
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