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Preparation And Storage
Recommended Assay Procedures
Recommended Assay Procedure:
Immunofluorescent staining and flow cytometry. The C11.5 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-12 producing cells within mixed cell populations. The FITC-, PE-, or APC-conjugated C11.5 antibodies are especially suitable for these studies (see figure). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/million cells). For specific methodology, please visit the protocols section or chapter on intracellular staining and flow cytometry in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.
A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the C11.5 antibody with ligand (e.g., recombinant human IL-12 p70, Cat. No. 554613 or recombinant human IL 12 p40, Cat. No. 554633) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled C11.5 antibody (Cat. No. 554573) prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is mouse IgG1 isotype controls, FITC-MOPC-21 (Cat. No. 554679); use control at comparable concentrations to antibody of interest.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Ficoll-Paque is a trademark of Amersham Biosciences Limited.
Companion Products
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.
Development References (4)
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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
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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
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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
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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: IC/FCM Block). View Reference
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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.
