PE Rat Anti-Mouse IL-12 (p40/p70)

BD Pharmingen™ PE Rat Anti-Mouse IL-12 (p40/p70)

Name: PE Rat Anti-Mouse IL-12 (p40/p70)
Brand: BD Pharmingen™
SKU: 562038

Clone C15.6

(RUO)

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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Product Details

Brand: BD Pharmingen™

Reactivity: Mouse (QC Testing)

Isotype: Rat IgG1

Immunogen: CHO-expressed recombinant mouse IL-12 p70 protein

Application: Intracellular staining (flow cytometry) (Routinely Tested)

Concentration: 0.2 mg/ml

RRID: AB_395420

Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.

Regulatory Status: RUO

Preparation And Storage

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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The C15.6 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-12 producing cells within mixed cell populations. The PE- and APC-conjugated C15.6 antibodies are especially suitable for these experiments (see figure). For optimal immunofluorescent staining and flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/million cells). For specific methodology, see The Immune Function Handbook at our website at www.bdbiosciences.com.

A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the PE-C15.6 antibody with excess ligand prior to staining, or 2) pre-block paraformaldehyde-fixed/saponin-permeabilized cells with unlabeled C15.6 antibody (e.g., Cat. No. 554477) 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 PE-R3-34 immunoglobulin (Cat. No. 554685); use at comparable concentrations to antibody of interest.

ELISA: The purified C15.6 antibody (Cat. No. 551219) is useful as a capture antibody for a sandwich ELISA for measuring mouse IL-12 p40 protein levels. The purified C15.6 antibody can be paired with the biotinylated C17.8 antibody (Cat. No. 554476) as the detection antibody, with recombinant mouse IL-12 p40 protein (Cat. No. 554594) as the standard. For testing mouse IL-12 p40 in complex biological fluids such as serum or plasma, our mouse IL-12 specific OptEIA™ sandwich ELISA set is recommended (Cat. No. 555165).

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.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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.

Companion Products

PE Rat IgG1, κ Isotype Control RUO

Size 0.1 mg Cat No. 554685

562038 Rev. 1

Antibody Details

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.

562038 Rev. 1

Format Details

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.

Format: PE

Excitation Source: Yellow-Green 488 nm, 532 nm, 561 nm

Excitation Max: 496 nm, 566 nm

Emission Max: 576 nm

562038 Rev.1

Citations & References

Development References (2)

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
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

562038 Rev. 1

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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.