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Alexa Fluor® 647 Rat Anti-Mouse IL-4
Alexa Fluor® 647 Rat Anti-Mouse IL-4

Expression of IL-4 by stimulated CD4+ and CD4-C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5,10 µg/ml, Cat. No.553726) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51,2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor® 647-11B11, Cat. No. 557739) (left panel) or immunoglobulin isotype control (Alexa Fluor® 647-R3-34, Cat. No. 557731) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor® 647 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL 4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining.  The quadarant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.

Expression of IL-4 by stimulated CD4+ and CD4-C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5,10 µg/ml, Cat. No.553726) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51,2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor® 647-11B11, Cat. No. 557739) (left panel) or immunoglobulin isotype control (Alexa Fluor® 647-R3-34, Cat. No. 557731) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor® 647 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL 4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining.  The quadarant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.

Product Details
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG1
Partially Purified Mouse IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_396846
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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

The conjugated 11B11 antibody can be used for multicolor 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 pretitrated. 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.

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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  7. 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.
557739 Rev. 3
Antibody Details
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11B11

Interleukin-4 (IL-4) is a pleiotropic cytokine that has many roles, such as inducing the differentiation of naïve helper T cells (Th0 cells) to Th2 cells, stimulating activated B-cell and T-cell proliferation, and promoting immunoglobulin class switching to IgG1 and IgE in mouse B-cells.  IL-4 is expressed by CD4 T-cells, mast cells, basophils and eosinophils.  IL-4 was previously known as B-Cell Differentiation Factor (BCDF) or B-cell Stimulatory Factor (BSF1).  The 11B11 monoclonal antibody specifically binds to mouse IL-4. The immunogen used to generate the 11B11 hybridoma was partially purified mouse IL-4 prepared from the supernatant of Phorbol 12-Myristate 13-Acetate (PMA)-stimulated EL-4 cells. The 11B11 antibody is reportedly 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.

557739 Rev. 3
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
557739 Rev.3
Citations & References
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Development References (4)

  1. Assenmacher M, Schmitz J, Radbruch A. Flow cytometric determination of cytokines in activated murine T helper lymphocytes: expression of interleukin-10 in interferon-gamma and in interleukin-4-expressing cells. Eur J Immunol. 1994; 24(5):1097-1101. (Clone-specific: Flow cytometry). View Reference
  2. Ohara J, Paul WE. Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1. Nature. 1985; 315(6017):333-336. (Immunogen). View Reference
  3. Sander B, Andersson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev. 1991; 119:65-93. (Clone-specific: ELISA, Flow cytometry). View Reference
  4. Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Clone-specific: ELISA, Flow cytometry, Neutralization). View Reference
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557739 Rev. 3

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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.