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554435Image1.png

Expression of IL-4 by stimulated CD4+ and CD4-BALB/c spleen cells. BALB/c spleen cells were cultured for 72 h in medium containing Staphylococcus aureus enterotoxin B (2 µg/ml; Sigma, St. Louis, MO), recombinant mouse IL-2 (10 U/ml, Cat. No. 550069) and recombinant mouse IL-4 (2 ng/ml, Cat. No. 550067). The cells were harvested and restimulated for 5 h with anti-CD3 (145-2C11, Cat. No. 553057 at 2 µg/ml) and anti-CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) antibodies in the presence of 3 µM monensin (BD GolgiStop, Cat No. 554704). The splenocytes were then stained with 0.25 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) and 0.25 µg of PE-conjugated rat anti-mouse IL-4 antibody (PE11B11, Cat. No 554435) by using the Pharmingen staining protocol (left panel). To demonstrate staining specificity, the binding of PE-11B11 was blocked by the preincubation of the conjugated antibody with excess recombinant mouse IL-4 (0.25 µg; Cat. No. 550067) (right panel) or by pre-blocking fixed/permeabilized cells with excess purified 11B11 mAb (5.0 µg; Cat. No. 554433) (data not shown), prior to staining. An approprate isotype control is PE-conjugated R3-34, (Cat. No. 554685). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified using the cytokine-blocking or mAb blocking controls.

18195A_554435_image2.png

Expression of IL-4 by stimulated CD4+ and CD4-BALB/c spleen cells. BALB/c spleen cells were cultured for 72 h in medium containing Staphylococcus aureus enterotoxin B (2 µg/ml; Sigma, St. Louis, MO), recombinant mouse IL-2 (10 U/ml, Cat. No. 550069) and recombinant mouse IL-4 (2 ng/ml, Cat. No. 550067). The cells were harvested and restimulated for 5 h with anti-CD3 (145-2C11, Cat. No. 553057 at 2 µg/ml) and anti-CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) antibodies in the presence of 3 µM monensin (BD GolgiStop, Cat No. 554704). The splenocytes were then stained with 0.25 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) and 0.25 µg of PE-conjugated rat anti-mouse IL-4 antibody (PE11B11, Cat. No 554435) by using the Pharmingen staining protocol (left panel). To demonstrate staining specificity, the binding of PE-11B11 was blocked by the preincubation of the conjugated antibody with excess recombinant mouse IL-4 (0.25 µg; Cat. No. 550067) (right panel) or by pre-blocking fixed/permeabilized cells with excess purified 11B11 mAb (5.0 µg; Cat. No. 554433) (data not shown), prior to staining. An approprate isotype control is PE-conjugated R3-34, (Cat. No. 554685). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified using the cytokine-blocking or mAb blocking controls.

554435Image1.png 18195A_554435_image2.png Red-Cap-Blue-Label.jpg

Expression of IL-4 by stimulated CD4+ and CD4-BALB/c spleen cells. BALB/c spleen cells were cultured for 72 h in medium containing Staphylococcus aureus enterotoxin B (2 µg/ml; Sigma, St. Louis, MO), recombinant mouse IL-2 (10 U/ml, Cat. No. 550069) and recombinant mouse IL-4 (2 ng/ml, Cat. No. 550067). The cells were harvested and restimulated for 5 h with anti-CD3 (145-2C11, Cat. No. 553057 at 2 µg/ml) and anti-CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) antibodies in the presence of 3 µM monensin (BD GolgiStop, Cat No. 554704). The splenocytes were then stained with 0.25 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) and 0.25 µg of PE-conjugated rat anti-mouse IL-4 antibody (PE11B11, Cat. No 554435) by using the Pharmingen staining protocol (left panel). To demonstrate staining specificity, the binding of PE-11B11 was blocked by the preincubation of the conjugated antibody with excess recombinant mouse IL-4 (0.25 µg; Cat. No. 550067) (right panel) or by pre-blocking fixed/permeabilized cells with excess purified 11B11 mAb (5.0 µg; Cat. No. 554433) (data not shown), prior to staining. An approprate isotype control is PE-conjugated R3-34, (Cat. No. 554685). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified using the cytokine-blocking or mAb blocking controls.

Expression of IL-4 by stimulated CD4+ and CD4-BALB/c spleen cells. BALB/c spleen cells were cultured for 72 h in medium containing Staphylococcus aureus enterotoxin B (2 µg/ml; Sigma, St. Louis, MO), recombinant mouse IL-2 (10 U/ml, Cat. No. 550069) and recombinant mouse IL-4 (2 ng/ml, Cat. No. 550067). The cells were harvested and restimulated for 5 h with anti-CD3 (145-2C11, Cat. No. 553057 at 2 µg/ml) and anti-CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) antibodies in the presence of 3 µM monensin (BD GolgiStop, Cat No. 554704). The splenocytes were then stained with 0.25 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) and 0.25 µg of PE-conjugated rat anti-mouse IL-4 antibody (PE11B11, Cat. No 554435) by using the Pharmingen staining protocol (left panel). To demonstrate staining specificity, the binding of PE-11B11 was blocked by the preincubation of the conjugated antibody with excess recombinant mouse IL-4 (0.25 µg; Cat. No. 550067) (right panel) or by pre-blocking fixed/permeabilized cells with excess purified 11B11 mAb (5.0 µg; Cat. No. 554433) (data not shown), prior to staining. An approprate isotype control is PE-conjugated R3-34, (Cat. No. 554685). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified using the cytokine-blocking or mAb blocking controls.

Product Details
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG1, κ
Partially Purified Mouse IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_395391
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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The antibody was conjugated to the dye under optimum conditions and unconjugated antibody and free dye were removed.
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Recommended Assay Procedures

Flow Cytometry: The 11B11 antibody has been reported to be useful for the immunofluorescent staining and flow cytometric analysis of IL-4-producing cells. The PE-conjugated 11B11 antibody can be used for multicolor flow cytometric analyses to identify and enumerate IL-4 producing cells within mixed cell populations (see right image). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be pretitrated (≤ 0.5 µg mAb/million cells).   An approprate isotype control is PE-conjugated R3-34, (Cat. No. 554685). For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining.

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 cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.

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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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. 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.
  5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
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562044 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.

562044 Rev. 3
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE 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 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
496 nm, 566 nm
576 nm
562044 Rev.3
Citations & References
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View product citations for antibody "562044" on CiteAb

Development References (9)

  1. Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
  2. 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
  3. Haak-Frendscho M, Brown JF, Iizawa Y, Wagner RD, Czuprynski CJ. Administration of anti-IL-4 monoclonal antibody 11B11 increases the resistance of mice to Listeria monocytogenes infection. J Immunol. 1992; 148(12):3978-3985. (Clone-specific: Neutralization). View Reference
  4. 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
  5. Openshaw P, Murphy EE, Hosken NA, et al. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J Exp Med. 1995; 182(5):1357-1367. (Clone-specific: Flow cytometry). View Reference
  6. 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). View Reference
  7. Sadick MD, Heinzel FP, Holaday BJ, Pu RT, Dawkins RS, Locksley RM. Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. Evidence for a T cell-dependent, interferon gamma-independent mechanism. J Exp Med. 1990; 171(1):115-127. (Clone-specific: Neutralization). View Reference
  8. 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
  9. 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
View All (9) View Less
562044 Rev. 3

Please refer to Support Documents for Quality Certificates

 

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.