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Cell Preparation and Separation Reagents
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- BD® Single-Cell Multiplexing Kit
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- BD Rhapsody™ TCR/BCR Profiling Assays
- BD® OMICS-Guard Sample Preservation Buffer
- BD Rhapsody™ ATAC-Seq Assays
- BD Rhapsody™ TCR/BCR Next Multiomic Assays
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Expression of IL-12 p40/p70 by activated CD14 + human PBMCs. Ficoll™-separated human PBMCs were primed for 2 hours with recombinant human IFN-γ (10 ng/ml final concentration; Cat. No. 554616), then activated with IFN-γ (10 ng/ml final concentration) and LPS (100 ng/ml final concentration; Sigma) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724) for an additional 22 hours. Cells were harvested, stained with PE-mouse anti-human CD14 antibody (Cat. No. 555398), fixed, permeabilized, and then stained with 0.125 µg of FITC-C11.5 antibody (Cat. No. 554574), following the BD Pharmingen 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 FITC-C11.5 antibody was blocked by preincubation of the conjugated antibody with recombinant human IL-12 p70 (Cat. No. 554613; middle panel) and by preincubation of the fixed/permeabilized cells with unlabeled C11.5 antibody (Cat. No. 554573; right panel) prior to staining with the FITC-C11.5 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
BD Pharmingen™ FITC Mouse Anti-Human IL-12 (p40/p70)
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推荐的实验流程
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.
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- 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.
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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.
研发参考 (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). 查看参考
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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). 查看参考
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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). 查看参考
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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). 查看参考
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