Two-color flow cytometric analysis of IL-4 expression in stimulated Mouse T cells. Mouse splenic CD4+ T cells were cultured (2 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 μg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml) antibodies with Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and Mouse IL-4 (Cat. No. 550067; 25 ng/ml) proteins. The cells were subsequently cultured (3 days) with Recombinant IL-2 and IL-4. The cells were then stimulated with Phorbol 12-Myristate 13-Acetate (PMA; Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) for 6 hours. The cells were washed and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The fixed cells were washed and then permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Rat Anti-Mouse CD4 antibody (Cat. No. 561091/553051) and with either BD Horizon™ RB613 Rat IgG1, κ Isotype Control (Cat. No. 571675; Left Plot) or BD Horizon™ RB613 Rat Anti-Mouse IL-4 antibody (Cat. No. 571206/571210; Right Plot) at 0.5 µg/test using the BD Biosciences Intracellular Cytokine Staining protocol. The bivariate pseudocolor density plot showing the correlated expression of IL-4 (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Two-color flow cytometric analysis of IL-4 expression in stimulated Mouse T cells. Mouse splenic CD4+ T cells were cultured (2 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 μg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml) antibodies with Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and Mouse IL-4 (Cat. No. 550067; 25 ng/ml) proteins. The cells were subsequently cultured (3 days) with Recombinant IL-2 and IL-4. The cells were then stimulated with Phorbol 12-Myristate 13-Acetate (PMA; Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) for 6 hours. The cells were washed and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The fixed cells were washed and then permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Rat Anti-Mouse CD4 antibody (Cat. No. 561091/553051) and with either BD Horizon™ RB613 Rat IgG1, κ Isotype Control (Cat. No. 571675; Left Plot) or BD Horizon™ RB613 Rat Anti-Mouse IL-4 antibody (Cat. No. 571206/571210; Right Plot) at 0.5 µg/test using the BD Biosciences Intracellular Cytokine Staining protocol. The bivariate pseudocolor density plot showing the correlated expression of IL-4 (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Product Details
BD Horizon™
IL4: Interleukin-4; BSF-1; B-cell growth factor 1; BCGF-1
Mouse (QC Testing)
Rat IgG1, κ
Partially Purified Mouse IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
16189
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 the dye under optimum conditions and unreacted dye was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures
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 cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
- 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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- CF™ is a trademark of Biotium, Inc.
- Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
571210 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.
571210 Rev. 1
Format Details
RB613
The BD Horizon RealBlue™ 613 (RB613) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 492-nm and an emission maximum (Em Max) at 613-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB613 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with reduced excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB613 can be used as an alternative to PE-CF594 and we recommend using an optical filter centered near 610-nm (eg, a 610/20-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-CF594. RB613 is on average brighter than PE-CF594 off the blue laser.
RB613
Blue 488 nm
492 nm
613 nm
571210 Rev.1
Citations & References
View product citations for antibody "571210" on CiteAb
Development References (10)
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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
571210 Rev. 1
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.