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PE Mouse Anti-Pig IFN-γ
PE Mouse Anti-Pig IFN-γ

Expression of IFN-γ by stimulated pig peripheral blood mononuclear cells (PBMC). Pig PBMC were stimulated for 20 hours with PMA (5 ng/ml final concentration, Sigma, Cat. No. P-8139) and ionomycin (500 ng/ml final concentration, Sigma, Cat. No. I-0634) in the presence of BD GolgiStop™ (4 µl/6 ml, Cat. No. 554724.). The PBMC were harvested, fixed, permeabilized, and subsequently stained with 0.015 µg of PE-conjugated mouse anti-porcine IFN-γ antibody (PE-P2G10, Cat. No. 559812) by using the BD Biosciences Pharmingen staining protocol (center panel). To demonstrate specificity of staining, the binding by the PE-P2G10 antibody was blocked by pre-incubation of the fixed/permeabilized cells with unlabeled P2G10 antibody (10 µg; Cat. No. 559961; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the staining profile using 0.12 µg of PE-MOPC-21 isotype control antibody (Cat. No. 554680, left panel) and verified using the unlabeled antibody blocking specificity control.

Expression of IFN-γ by stimulated pig peripheral blood mononuclear cells (PBMC). Pig PBMC were stimulated for 20 hours with PMA (5 ng/ml final concentration, Sigma, Cat. No. P-8139) and ionomycin (500 ng/ml final concentration, Sigma, Cat. No. I-0634) in the presence of BD GolgiStop™ (4 µl/6 ml, Cat. No. 554724.). The PBMC were harvested, fixed, permeabilized, and subsequently stained with 0.015 µg of PE-conjugated mouse anti-porcine IFN-γ antibody (PE-P2G10, Cat. No. 559812) by using the BD Biosciences Pharmingen staining protocol (center panel). To demonstrate specificity of staining, the binding by the PE-P2G10 antibody was blocked by pre-incubation of the fixed/permeabilized cells with unlabeled P2G10 antibody (10 µg; Cat. No. 559961; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the staining profile using 0.12 µg of PE-MOPC-21 isotype control antibody (Cat. No. 554680, left panel) and verified using the unlabeled antibody blocking specificity control.

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BD Pharmingen™
Pig (QC Testing)
Mouse IgG1, κ
Recombinant pig IFN-γ protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_397341
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. 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.

推奨アッセイ手順

IF/Flow: For immunofluorescent staining and flow cytometric analysis, the P2G10 antibody has been found useful to identify and enumerate IFN-γ producing cells within mixed cell populations. PE- conjugated P2G10 antibody (Cat. No. 559812) is especially suitable for these studies. For specific methodology, please visit the online protocols section or chapter on intracellular staining 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 fluorochrome-conjugated P2G10 antibody with ligand prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled P2G10 antibody (Cat. No. 559961) 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, κ isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized porcine cells is PE-MOPC-21 immunoglobulin (Cat. No. 554680); use at comparable concentrations to antibody of interest (eg, ≤ 0.5 µg mAb/1 million cells).

ELISA Capture: The purified P2G10 antibody (Cat. No. 559961) is useful as a capture antibody for a sandwich ELISA for measuring porcine IFN-γ protein levels. Purified P2G10 antibody can be paired with the biotinylated P2C11 antibody (Cat. No. 559958) as the detecting antibody.

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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. 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.
559812 Rev. 1
抗体の詳細
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P2G10

The P2G10 monoclonal antibody specifically binds to porcine interferon-γ (IFN-γ). The immunogen used to generate the P2G10 hybridoma was recombinant pig IFN-γ protein.

559812 Rev. 1
フォーマットの詳細
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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.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
559812 Rev.1
引用&参考文献
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Development References (2)

  1. Mateu de Antonio E, Husmann RJ, Hansen R, et al. Quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen. Vet Immunol Immunopathol. 1998; 61(2-4):265-277. (Immunogen: ELISA). View Reference
  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
559812 Rev. 1

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.