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V450 Rat Anti-Mouse IL-4
V450 Rat Anti-Mouse IL-4

Flow cytometric analysis for IL-4 in activated mouse splenocytes.  Mouse Intracellular Cytokine-2 positive control cells (MiCK-2) offered by BD Biosciences as MN 554653, are activated mouse splenocytes prepared in the presence of a protein transport inhibitor.  Fixed and permeabilized MiCK-2 cells were stained either with a BD Horizon™ V450 Rat IgG1, κ isotype control (left panel) or with the BD Horizon™ V450 Rat Anti-Mouse IL-4 antibody (right panel).  Dot plots were derived from gated events based on light scattering characteristics for lymphocytes.  Flow cytometry was performed on a BD LSR™ II flow cytometry system.

Flow cytometric analysis for IL-4 in activated mouse splenocytes.  Mouse Intracellular Cytokine-2 positive control cells (MiCK-2) offered by BD Biosciences as MN 554653, are activated mouse splenocytes prepared in the presence of a protein transport inhibitor.  Fixed and permeabilized MiCK-2 cells were stained either with a BD Horizon™ V450 Rat IgG1, κ isotype control (left panel) or with the BD Horizon™ V450 Rat Anti-Mouse IL-4 antibody (right panel).  Dot plots were derived from gated events based on light scattering characteristics for lymphocytes.  Flow cytometry was performed on a BD LSR™ II flow cytometry system.

Product Details
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BD Horizon™
Mouse (QC Testing)
Rat IgG1
Partially Purified Mouse IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_1727550
Aqueous buffered solution containing protein stabilizer 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™ V450 under optimum conditions, and unreacted BD Horizon™ V450 was removed.

Recommended Assay Procedures

Flow cytometry: The 11B11 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-4 producing cells within mixed cell populations.  A useful control investigators may consider using for demonstrating specificity of staining, is to pre-block with one of the following reagents: (1) recombinant mouse IL-4 (Cat. No. 550067) or (2) unlabeled 11B11 antibody (Cat. No. 554434), prior to staining.

Cell Preparation:  Investigators not wishing to utilize MiCK-2 cells may alternatively stimulate mouse splenocyte enriched CD4+ cells (e.g C57BL/6) with 10 µg/ml plate-bound NA/LE hamster anti-mouse CD3e antibody (clone 145-2C11; Cat. No. 553057) and 2 µg/ml soluble NA/LE hamster anti-mouse CD28 (clone 37.51; Cat. No. 553294) antibody in the presence of 10 ng/ml recombinant mouse IL-2 (Cat. No. 550069) and 20 ng/ml recombinant mouse IL-4 (Cat. No. 550067) for 2 days followed by additional cell expansion with recombinant IL-2 and IL-4 for an additional 3 days. Following expansion, cells may be activated with the Leukocyte Activation Cocktail (Cat. No. 550583) or alternatively, with a 4-6 hr treatment with PMA (5 ng/mL, Sigma-Aldrich Cat. No. P-8139) and ionomycin (500 ng/mL, Sigma-Aldrich Cat. No. I-0634) in the presence of 1 µg/mL Brefeldin A (BD GolgiPlug™ MN 555029).  Investigators are advised to fix and permeabilize the cells prior to staining.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. BD Horizon V450 has a maximum absorption of 406 nm and maximum emission of 450 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
  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. Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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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.

The antibody is conjugated to BD Horizon™ V450, which has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser Ex max of 406 nm and has an Em Max at 450 nm. Conjugates with BD Horizon™ V450 can be used in place of Pacific Blue™ conjugates.

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Format Details
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V450
BD Horizon™ V450 Dye is part of the BD Horizon™ violet family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 405-nm and an emission maximum (Em Max) at 450-nm. BD Horizon™ V450, driven by BD innovation, is designed to be excited by the violet laser (405 nm) and detected using an optical filter centered near 450-nm (e.g., a 450/50-nm bandpass filter). The dye can be excited by the UV (355-nm) laser resulting in cross-laser excitation and spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
V450
Violet 405 nm
405 nm
450 nm
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Citations & References
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Development References (17)

  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. (Biology). View Reference
  2. Bogen SA, Fogelman I, Abbas AK. Analysis of IL-2, IL-4, and IFN-gamma-producing cells in situ during immune responses to protein antigens. J Immunol. 1993; 150(10):4197-4205. (Biology). View Reference
  3. Chatelain R, Varkila K, Coffman RL. IL-4 induces a Th2 response in Leishmania major-infected mice. J Immunol. 1992; 148(4):1182-1187. (Biology). View Reference
  4. Finkelman FD, Madden KB, Morris SC, et al. Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine-anti-cytokine antibody complexes. J Immunol. 1993; 151(3):1235-1244. (Biology). View Reference
  5. Fujihashi K, McGhee JR, Beagley KW, et al. Cytokine-specific ELISPOT assay. Single cell analysis of IL-2, IL-4 and IL-6 producing cells. J Immunol Methods. 1993; 160(2):181-189. (Biology). View Reference
  6. Gillis S, Ferm MM, Ou W, Smith KA. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978; 120(6):2027-2032. (Biology). View Reference
  7. 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. (Biology). View Reference
  8. Helms T, Boehm BO, Asaad RJ, Trezza RP, Lehmann PV, Tary-Lehmann M. Direct visualization of cytokine-producing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J Immunol. 2000; 164(7):3723-3732. (Biology). View Reference
  9. Klinman D and Nutman T. ELISPOT assay to detect cytokine-secreting murine and human cells. In: Coligan J, Kruisbeek A, Margulies D, Shevach E, Strober W, ed. Current Protocols in Immunology. 1994:6-19.
  10. 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. (Biology). View Reference
  11. Litton MJ, Sander B, Murphy E, O'Garra A, Abrams JS. Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J Immunol Methods. 1994; 175(1):47-58. (Biology). View Reference
  12. Paul WE. Interleukin-4: a prototypic immunoregulatory lymphokine. Blood. 1991; 77(9):1859-1870. (Biology). View Reference
  13. 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: Flow cytometry). View Reference
  14. 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. (Biology). View Reference
  15. 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. (Biology). View Reference
  16. Shirai A, Sierra V, Kelly CI, Klinman DM. Individual cells simultaneously produce both IL-4 and IL-6 in vivo. Cytokine. 1994; 6(3):329-336. (Biology). View Reference
  17. 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. (Biology). View Reference
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