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PE Rat Anti-Mouse IL-12 (p40/p70)
PE Rat Anti-Mouse IL-12 (p40/p70)
Expression of IL-12 by mouse bone marrow-derived macrophages. Bone marrow cells from 6 month old BALB/c mice were cultured for 10 days in mouse GM-CSF (40 ng/ml final concentration; Cat. No. 554586). Adherent cells were washed and treated for ~14 hours with mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587); subsequently LPS (1 µg/ml final concentration; Sigma) and GolgiStop™ (2 µM final concentration; Cat. No. 554724) were added to cultures for an additional 5 hours. Adherent cells were washed and then incubated with 1x trypsin EDTA at 37°C for 15 minutes and gently dislodged by pipetting. Nonspecific surface binding was blocked by incubation of cells with purified polyclonal normal mouse immunoglobulin. Cells were then fixed, permeabilized, and non-specific binding to intracellular antigens was blocked using BD Cytofix/Cytoperm™ (Cat. No. 554714). Cells were then stained with 0.06 µg of PE-conjugated rat anti-mouse IL-12 (p40/p70) antibody (PE-C15.6, Cat. No. 554479; see left panel) using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of PE-C15.6 antibody was blocked by the preincubation of the fixed/permeabilized cells with unlabeled C15.6 antibody (5 µg/ml final concentration; Cat. No. 554477; see right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified using the ligand blocking and unlabeled antibody blocking controls.
Expression of IL-12 by mouse bone marrow-derived macrophages. Bone marrow cells from 6 month old BALB/c mice were cultured for 10 days in mouse GM-CSF (40 ng/ml final concentration; Cat. No. 554586). Adherent cells were washed and treated for ~14 hours with mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587); subsequently LPS (1 µg/ml final concentration; Sigma) and GolgiStop™ (2 µM final concentration; Cat. No. 554724) were added to cultures for an additional 5 hours. Adherent cells were washed and then incubated with 1x trypsin EDTA at 37°C for 15 minutes and gently dislodged by pipetting. Nonspecific surface binding was blocked by incubation of cells with purified polyclonal normal mouse immunoglobulin. Cells were then fixed, permeabilized, and non-specific binding to intracellular antigens was blocked using BD Cytofix/Cytoperm™ (Cat. No. 554714). Cells were then stained with 0.06 µg of PE-conjugated rat anti-mouse IL-12 (p40/p70) antibody (PE-C15.6, Cat. No. 554479; see left panel) using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of PE-C15.6 antibody was blocked by the preincubation of the fixed/permeabilized cells with unlabeled C15.6 antibody (5 µg/ml final concentration; Cat. No. 554477; see right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified using the ligand blocking and unlabeled antibody blocking controls.
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Antibody Details
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C15.6

The C15.6 monoclonal antibody specifically binds to both free and complexed (homodimer p80 and heterodimer p70) forms of the p40 subunit of mouse interleukin-12 (IL-12). The immunogen used to generate the C15.6 hybridoma was recombinant mouse IL-12 p70 protein. p40 has also been described as a subunit of IL-23 and thus it is possible that the C15.6 antibody will crossreact with IL-23.

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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.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
Citations & References
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Development References (2)

  1. 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
  2. Wysocka M, Kubin M, Vieira LQ, et al. Interleukin-12 is required for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice. Eur J Immunol. 1995; 25(3):672-676. (Clone-specific: ELISA). View Reference

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