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    PE Rat Anti-Mouse IFN-γ
    PE Rat Anti-Mouse IFN-γ
    Expression of IFN-γ by stimulated CD8+ and CD8- BALB/c spleen cells. Splenocytes from 6 month old BALB/c mice were cultured for 3 days in the presence of SEB (2 µg/ml; Sigma, Cat. No. S-4881), then restimulated for 5 hour with hamster anti-mouse CD3 (2 µg/ml, 145-2C11, Cat. No. 553057) and hamster anti-mouse CD28 (2 µg/ml, 37.51, Cat. No, 553294) antibodies in the presence of 2 µM GolgiStop™ (aka, monensin; Cat. No. 554724). The splenocytes were harvested, stained with 0.06 µg of FITC rat anti-mouse CD8 (FITC 53-6.7, Cat. No. 553030), fixed, permeabilized, and subsequently stained with 0.06 µg of PE rat anti-mouse IFN-γ (PE-XMG1.2, Cat. No. 554412, left panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding by the PE-XMG1.2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled XMG1.2 antibody (5.0 µg; see right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
    PE Rat Anti-Mouse IFN-γ
    Expression of IFN-γ by stimulated CD8+ and CD8- BALB/c spleen cells. Splenocytes from 6 month old BALB/c mice were cultured for 3 days in the presence of SEB (2 µg/ml; Sigma, Cat. No. S-4881), then restimulated for 5 hour with hamster anti-mouse CD3 (2 µg/ml, 145-2C11, Cat. No. 553057) and hamster anti-mouse CD28 (2 µg/ml, 37.51, Cat. No, 553294) antibodies in the presence of 2 µM GolgiStop™ (aka, monensin; Cat. No. 554724). The splenocytes were harvested, stained with 0.06 µg of FITC rat anti-mouse CD8 (FITC 53-6.7, Cat. No. 553030), fixed, permeabilized, and subsequently stained with 0.06 µg of PE rat anti-mouse IFN-γ (PE-XMG1.2, Cat. No. 554412, left panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding by the PE-XMG1.2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled XMG1.2 antibody (5.0 µg; see right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
    Product Details
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    BD Pharmingen™
    Mouse (QC Testing)
    Rat IgG1, κ
    Mouse IFN-γ Recombinant Protein
    Intracellular staining (flow cytometry) (Routinely Tested)
    0.2 mg/ml
    15978
    AB_395376
    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.
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    Recommended Assay Procedures

    Immunofluorescent Staining and Flow Cytometric Analysis: The XMG1.2 antibody is useful for the immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. The PE-conjugated XMG1.2 antibody is especially suitable for these studies. For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/million cells). A useful control for demonstrating specificity of staining is either of the following: (1) pre-block the fluorochrome-conjugated XMG1.2 antibody prior to staining, with unlabeled XMG1.2 antibody (Cat. No. 554409), or (2) pre-block with a molar excess of ligand, (e.g. recombinant mouse IFN-g; Cat. No. 554587) prior to staining.

    A suitable rat IgG1 isotype control 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.

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    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.
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    562020 Rev. 1
    Antibody Details
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    XMG1.2

    The XMG1.2 monoclonal antibody specifically binds to mouse interferon-γ (IFN-γ) protein. IFN-γ is a pleiotropic cytokine, of approximately 15-17 kDa, involved in the regulation of inflammatory and immune responses. It plays an important role in activation, growth, and differentiation of T and B lymphocytes, macrophages, NK cells and other non-hematopoietic cell types. IFN-γ production is associated with the Th1 cell differentiation. The purified form of this antibody has been reported to be a neutralizing antibody.

    562020 Rev. 1
    Format Details
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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
    562020 Rev.1
    Citations & References
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    Development References (4)

    1. Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific). View Reference
    2. Cherwinski HM, Schumacher JH, Brown KD, Mosmann TR. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J Exp Med. 1987; 166(5):1229-1244. (Clone-specific). View Reference
    3. 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: Blocking, Neutralization). View Reference
    4. Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Clone-specific). View Reference
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    562020 Rev. 1

    Please refer to Support Documents for Quality Certificates


    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.

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