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      Purified Mouse Anti-Human IL-12 (p40/p70)
      Purified Mouse Anti-Human IL-12 (p40/p70)
      Expression of IL-12 p40/p70 by activated CD14+ human PBMCs. Ficoll-separated human PBMCs were primed for 2 hrs with recombinant human IFN-γ (10 ng/ml; Cat. No. 554616), then activated with IFN-γ (10 ng/ml) and LPS (100 ng/ml; Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724) for an additional 22 hr. Cells were harvested, stained with FITC-mouse anti human CD14 antibody (Cat. No. 555397), fixed, permeabilized, and then stained with 0.125 µg of PE-C11.5 antibody (Cat. No. 554575), following Pharmingen's staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding of the PE-C11.5 antibody was blocked by preincubation of the fixed/permeabilized cells with the unlabeled C11.5 antibody (5 µg, Cat. No. 554573) prior to staining (right panel). 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.  
      Purified Mouse Anti-Human IL-12 (p40/p70)
      Expression of IL-12 p40/p70 by activated CD14+ human PBMCs. Ficoll-separated human PBMCs were primed for 2 hrs with recombinant human IFN-γ (10 ng/ml; Cat. No. 554616), then activated with IFN-γ (10 ng/ml) and LPS (100 ng/ml; Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724) for an additional 22 hr. Cells were harvested, stained with FITC-mouse anti human CD14 antibody (Cat. No. 555397), fixed, permeabilized, and then stained with 0.125 µg of PE-C11.5 antibody (Cat. No. 554575), following Pharmingen's staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding of the PE-C11.5 antibody was blocked by preincubation of the fixed/permeabilized cells with the unlabeled C11.5 antibody (5 µg, Cat. No. 554573) prior to staining (right panel). 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™
      Human (QC Testing)
      Mouse IgG1
      CHO-expressed recombinant human IL-12 p70 heterodimer
      Intracellular block/flow cytometry (Routinely Tested)
      0.5 mg/ml
      AB_398573
      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.
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      Recommended Assay Procedures

      Recommended Assay Procedure:

      Blocking Control for Intracellular Staining: The purified C11.5 antibody can be used as a blocking control to demonstrate specificity of IL-12 staining by PE-C11.5, FITC-C11.5 and APC-C11.5 antibodies (Cat. No. 554575, No. 554574, No. 554576). To perform this control, the fixed/permeabilized cells (~ 1 million) can be incubated with 1 -10 µg of unlabeled C11.5 antibody (Cat. No. 554573) for 20 minutes at 4°C, prior to staining with PE-C11.5, FITC-C11.5 or APC-C11.5 antibodies (e.g., 0.1 -0.5 µg mAb/1 million cells). The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. 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.

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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. 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.
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      554573 Rev. 1
      Antibody Details
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      C11.5

      The C11.5 monoclonal antibody specifically binds to the human IL-12 p40 monomer and p70 heterodimer, but does not bind to the IL-12 p35 monomer. The immunogen used to generate the C11.5 hybridoma was the CHO-expressed recombinant human IL-12 p70 heterodimer.  p40 has also been described as a subunit of IL-23 and thus it is possible that the C11.5 antibody crossreacts with IL-23.

      This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.

      554573 Rev. 1
      Format Details
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      Purified
      Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
      Purified
      554573 Rev.1
      Citations & References
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      Development References (4)

      1. D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993; 178(3):1041-1048. (Clone-specific). View Reference
      2. D'Andrea A, Rengaraju M, Valiante NM, et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J Exp Med. 1992; 176(5):1387-1398. (Clone-specific). View Reference
      3. Oppmann B, Lesley R, Blom B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.. Immunity. 2000; 13(5):715-25. (Biology). View Reference
      4. 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
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      554573 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.

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