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Expression of IFN-γ by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (50 ng/ml; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (250 ng/ml; Sigma, Cat. No. C-9275) in the presence of GolgiStop™ (Cat. No. 554724). The PBMC were stained with PE-Cy™5 Mouse Anti-Human CD3 (Cat. No. 555334/561007), fixed and permeabilized with BD Cytofix/Cytoperm Plus Kit (with BD GolgiStop) (Cat. No. 554715), and subsequently stained with 0.25 µg of PE Mouse Anti-Human IFN-γ (Cat. No. 554701/559327). To demonstrate staining specificity, PE Mouse Anti-Human IFN-γ antibody binding was blocked by preincubation of fixed/permeabilized cells with excess Purified Mouse Anti-Human IFN-γ (5 µg; Cat. No. 554699/550011; right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on autofluorescence controls and verified using the unlabeled antibody blocking control.
BD Pharmingen™ Purified Mouse Anti-Human IFN-γ
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
Blocking Control for Intracellular Staining: The purified B27 antibody can be used as a blocking control to demonstrate specificity of IFN-γ
staining by PE-B27 antibody or FITC-B27 antibody (Cat. No. 554701/554700). To perform this control, the fixed/permeabilized cells (~1
million) can be incubated with 1-10 µg of unlabeled B27 antibody (Cat. No. 554669) for 20 minutes at 4°C, prior to staining with PE-B27
antibody or FITC-B27 antibody (e.g., 0.1-0.5 µg mAb/1 million cells). The intracellular cytokine staining technique and use of blocking controls
are described in detail by C. Prussin and D. Metcalfe.
Neutralization: The NA/LE™ B27 antibody is useful for neutralization of human IFN-γ bioactivity. This purified B27 antibody (Cat. No. 554698) is supplied in sodium azide free, sterile-filtered (0.22 µm pore) PBS, pH 7.2. Endotoxin level as determined by LAL assay is less than 0.01 ng/µg protein.
IP/WB: The B27 antibody has been reported to be useful for immunoprecipitation studies. The B27 antibody has been reported not to bind to denatured IFN-γ.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The B27 monoclonal antibody specifically binds to human interferon-γ (IFN-γ), a 14-18 kDa glycoprotein containing 143 amino acid residues. IFN-γ is a potent multifunctional cytokine produced by several activated cell types including NK, NKT, CD4+TCRαβ+, CD8+TCRαβ+, and TCRγδ+ T cells. IFN-γ exerts its biological effects through specific binding to the high-affinity IFN-γ receptor complex comprised of IFN-γRα (CD119) and IFN-γRβ subunits. In addition to its antiviral effects, IFN-γ upregulates a number of lymphoid cell functions including the antimicrobial and anti-tumor responses of macrophages, NK cells, and neutrophils. In addition, IFN-γ influences the regulation of proliferation, differentiation, and effector responses of B cell and T cell subsets. These influences can involve IFN-γ's capacity to boost MHC class I and II expression by antigen-presenting cells as well as direct effects on B cells and T cells themselves. B27 is a neutralizing antibody. The use of B27 antibody for epitope mapping of human IFN-γ has been described. The B27 antibody has been reported not to bind to denatured IFN-γ.
Development References (5)
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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
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Favre C, Wijdenes J, Cabrillat H, Djossou O, Banchereau J, de Vries JE. Epitope mapping of recombinant human gamma interferon using monoclonal antibodies. Mol Immunol. 1989; 26(1):17-25. (Clone-specific: Immunoprecipitation, Neutralization, Western blot). View Reference
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Fonteneau JF, Le Drean E, Le Guiner S, Gervois N, Diez E, Jotereau F. Heterogeneity of biologic responses of melanoma-specific CTL. J Immunol. 1997; 159(6):2831-2839. (Biology). View Reference
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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). View Reference
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Rotteveel FT, Kokkelink I, van Lier RA, et al. Clonal analysis of functionally distinct human CD4+ T cell subsets. J Exp Med. 1988; 168(5):1659-1673. (Biology). View Reference
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.