Flow cytometric analysis of IL-4 expressed in activated mouse splenocytes. Splenocytes from C57BL/6 mice were enriched for CD4+ T cells by positive selection using Purified NA/LE Rat Anti-Mouse CD4 antibody-coated plates (GK1.5, Cat. No.553726;10 μg/ml) for 1 hr at 4°C. The CD4+ T cells were harvested and stimulated with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (145-2C11, Cat. No. 553057;10 μg/ml) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (37.51, Cat. No. 553294; 2 μg/ml) antibody and Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 50 ng/ml) for 2 days. The cells were expanded in IL-2 and IL-4 for 3 days and then washed and stimulated (4 hr) with PMA (Sigma, Cat. No. P-8139; 5 ng/ml) and ionomycin (Sigma, Cat. No. P-8139; 500 ng) in the presence of BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029). The activated cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized using the BD Perm/Wash™ Permeabilization Buffer (Cat. No. 554723) and then stained either with a BD Horizon™ BV421 Rat IgG1, κ Isotype Control (Cat. No. 562868, Left Panel) or with BD Horizon™ BV421 Rat Anti-Mouse IL-4 antibody (Cat. No. 562915, Right Panel). Two-color dot plots showing IL-4 (or Ig isotype control staining) versus autofluorescence were derived from gated events with the forward and light scattering characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSR™ II Flow Cytometry System.
Flow cytometric analysis of IL-4 expressed in activated mouse splenocytes. Splenocytes from C57BL/6 mice were enriched for CD4+ T cells by positive selection using Purified NA/LE Rat Anti-Mouse CD4 antibody-coated plates (GK1.5, Cat. No.553726;10 μg/ml) for 1 hr at 4°C. The CD4+ T cells were harvested and stimulated with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (145-2C11, Cat. No. 553057;10 μg/ml) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (37.51, Cat. No. 553294; 2 μg/ml) antibody and Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 50 ng/ml) for 2 days. The cells were expanded in IL-2 and IL-4 for 3 days and then washed and stimulated (4 hr) with PMA (Sigma, Cat. No. P-8139; 5 ng/ml) and ionomycin (Sigma, Cat. No. P-8139; 500 ng) in the presence of BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029). The activated cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized using the BD Perm/Wash™ Permeabilization Buffer (Cat. No. 554723) and then stained either with a BD Horizon™ BV421 Rat IgG1, κ Isotype Control (Cat. No. 562868, Left Panel) or with BD Horizon™ BV421 Rat Anti-Mouse IL-4 antibody (Cat. No. 562915, Right Panel). Two-color dot plots showing IL-4 (or Ig isotype control staining) versus autofluorescence were derived from gated events with the forward and light scattering characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSR™ II Flow Cytometry System.
Product Details
BD Horizon™
IL4: Interleukin-4; BSF-1; B-cell growth factor 1; BCGF-1
Mouse (QC Testing)
Rat IgG1
Partially Purified Mouse IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_2737889
Aqueous buffered solution containing BSA and ≤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 BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 were removed.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Brilliant Violet™ 421 is a trademark of Sirigen.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
BV421 Rat IgG1, κ Isotype Control RUO
Size
50 µg
Cat No.
562868
Purified NA/LE Rat Anti-Mouse CD4 RUO
Size
0.5 mg
Cat No.
553726
Purified NA/LE Hamster Anti-Mouse CD3e RUO
Size
0.5 mg
Cat No.
553057
566288 Rev. 1
Antibody Details
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.
The antibody was conjugated to BD Horizon™ BV421 which is part of the BD Horizon™ Brilliant Violet™ family of dyes. With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon™ BV421 can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon™ BV421 conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue™ conjugates.
566288 Rev. 1
Format Details
BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
BV421
Violet 405 nm
407 nm
423 nm
566288 Rev.1
Citations & References
Development References (10)
-
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
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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
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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
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Lindqvist C, Lundstrom H, Oker-Blom C, Akerman KE. Enhanced IL-4-mediated D10.G4.1 proliferation with suboptimal concentrations of anti-IL-4 receptor monoclonal antibodies. J Immunol. 1993; 150(2):394-398. (Clone-specific: Neutralization). View Reference
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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
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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
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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
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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
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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). View Reference
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Swain SL, Weinberg AD, English M, Huston G. IL-4 directs the development of Th2-like helper effectors. J Immunol. 1990; 145(11):3796-3806. (Clone-specific: Neutralization). View Reference
566288 Rev. 1
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.
