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      PE Rat Anti-Human IL-13
      PE Rat Anti-Human IL-13
      Expression of IL-13 by stimulated human CD4+ cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 antibody (UCHT1, 10 µg/ml for plate coating, Cat. No. 555329), soluble anti-human CD28 antibody (20 ng/ml, Cat. No. 555725), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and recombinant human IL-4 (10 ng/ml, Cat. No. 554605) for 2 days. The cells were washed and subsequently cultured in medium containing IL-2 and IL-4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hr with PMA (Sigma, Cat. #P-8139; 50 ng/ml), calcium ionophore A23187 (Sigma, Cat. #C-9275; 250 ng/ml) in the presence of BD GolgiStop™ (2 µM final concentration, Cat. No. 554724). The cells were stained with PE-Cy5-anti-CD4 (PE-CY5-RPA-T4, Cat. No. 555348) fixed, permeabilized, and subsequently stained with 20 µl of PE-rat anti-human IL-13 antibody (PE-JES10-5A2; Cat. No. 559328) by following the Usage section below (see Right Figure, left panel). To demonstrate specificity of staining, the binding of PE-JES10-5A2 antibody was blocked by the preincubation of the conjugated antibody with excess recombinant human IL-13 (see middle panel), and by preincubation of the fixed/permeabilized cells with unlabeled JES10-5A2 antibody (5 µg, Cat. No. 554570; see right panel) prior to staining with the PE-JES10-5A2 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified with the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
      PE Rat Anti-Human IL-13
      Expression of IL-13 by stimulated human CD4+ cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 antibody (UCHT1, 10 µg/ml for plate coating, Cat. No. 555329), soluble anti-human CD28 antibody (20 ng/ml, Cat. No. 555725), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and recombinant human IL-4 (10 ng/ml, Cat. No. 554605) for 2 days. The cells were washed and subsequently cultured in medium containing IL-2 and IL-4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hr with PMA (Sigma, Cat. #P-8139; 50 ng/ml), calcium ionophore A23187 (Sigma, Cat. #C-9275; 250 ng/ml) in the presence of BD GolgiStop™ (2 µM final concentration, Cat. No. 554724). The cells were stained with PE-Cy5-anti-CD4 (PE-CY5-RPA-T4, Cat. No. 555348) fixed, permeabilized, and subsequently stained with 20 µl of PE-rat anti-human IL-13 antibody (PE-JES10-5A2; Cat. No. 559328) by following the Usage section below (see Right Figure, left panel). To demonstrate specificity of staining, the binding of PE-JES10-5A2 antibody was blocked by the preincubation of the conjugated antibody with excess recombinant human IL-13 (see middle panel), and by preincubation of the fixed/permeabilized cells with unlabeled JES10-5A2 antibody (5 µg, Cat. No. 554570; see right panel) prior to staining with the PE-JES10-5A2 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified with the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
      Product Details
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      BD Pharmingen™
      Interleukin-13; NC30; ALRH; BHR1; P600
      Human (QC Testing)
      Rat IgG1
      Human Recombinant IL-13
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      3596
      AB_397225
      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 R-PE under optimum conditions, and unconjugated antibody and free PE were removed.
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      Recommended Assay Procedures

      Immunofluorescent Staining and Flow Cytometric Analysis: The PE-conjugated JES10-5A2 antibody can be used for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-13 producing cells within mixed cell populations (see Figure). This 100 Test Size formulation of the PE-conjugated JES10-5A2 antibody has been pre-titrated to assure effective intracellular detection of human IL-13 using 20 µl/1 x 10^6 cells. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook.

      A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the PE-JES10-5A2 antibody with ligand (e.g., human IL-13) prior to staining, or 2) pre-block paraformaldehyde-fixed/saponin-permeabilized cells with unlabeled JES10-5A2 antibody (e.g., Cat. No. 554570) prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable rat IgG1 isotype control immunoglobulin for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse or human cells is also available in a 100 Test Size formulation PE-R3-34 antibody (Cat. No. 559318).

      Important Note: This pre-titered antibody solution does not contain a cell permeabilization agent. It is necessary to include a cell permeabilization agent when using the pre-titered antibody solution to stain fixed and permeabilized cells. Perm/Wash™ Buffer (Cat. No. 554723) contains the permeabilization agent saponin and is useful for this purpose as described in the Usage section below.

      Usage

      1. Resuspend 1 x 10/^6 fixed and permeabilized cells in 20 µl of the pre-titered antibody solution and 30 µl of 1X Perm/Wash™ Buffer (Cat. No. 554723).

      2. Incubate the cell suspension for 15 minutes (at RT or 4°C).

      3. Wash twice in 100 µl of 1X Perm/Wash™ Buffer (Cat. No. 554723).

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      Product Notices

      1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
      2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      5. 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.
      6. An isotype control should be used at the same concentration as the antibody of interest.
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      559328 Rev. 2
      Antibody Details
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      JES10-5A2

      The JES10-5A2 monoclonal antibody specifically binds to human interleukin-13, IL-13. IL-13 is produced by activated T cells, mast cells and NK cells. IL-13 regulates IgE production by B cells and can suppress the cytotoxic activity of macrophages and their production of inflammatory mediators. The immunogen used to produce the JES10-5A2 hybridoma was COS-expressed recombinant human IL-13. This is a neutralizing antibody.

      559328 Rev. 2
      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.
      altImg
      PE
      Yellow-Green 488 nm, 532 nm, 561 nm
      496 nm, 566 nm
      576 nm
      559328 Rev.2
      Citations & References
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      View product citations for antibody "559328" on CiteAb

      Development References (3)

      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. McKenzie A, Zurawski G. Measurement of IL-13. In: Coligan, Kruisbeek, Shevak, Strober, ed. Current Protocols in Immunology. New York: John Wiley & Sons; 1994:18-19.
      3. 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: IC/FCM Block). View Reference
      559328 Rev. 2

       

      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.