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FITC Mouse Anti-Human IFN-γ
FITC Mouse Anti-Human IFN-γ
Expression of IFN-γ by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (250 ng/ml final concentration; Sigma, Cat. No. C-9275) in the presence of GolgiStop™ (Cat. No. 554724). The PBMC were stained with PE-Cy5 anti-CD3 (Cat. No. 555334/561007), fixed and permeabilized with BD Cytofix/Cytoperm Plus Kit (with BD GolgiStop) (Cat. No. 554715), then subsequently stained with 0.25 µg of FITC Mouse Anti-Human IFN-γ antibody (Cat. No. 554700/552887/561053; left panel). To demonstrate specificity of staining, binding by the FITC Mouse Anti-Human IFN-γ antibody was blocked by preincubation of fixed/permeabilized cells with excess Purified Mouse Anti-Human IFN-γ antibody (Cat. No. 554699/550011, right panel). The quadrant markers for the bivariate dot plot were set based on autofluorescence controls, and verified using the unlabeled B27 antibody blocking control.
Expression of IFN-γ by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (250 ng/ml final concentration; Sigma, Cat. No. C-9275) in the presence of GolgiStop™ (Cat. No. 554724). The PBMC were stained with PE-Cy5 anti-CD3 (Cat. No. 555334/561007), fixed and permeabilized with BD Cytofix/Cytoperm Plus Kit (with BD GolgiStop) (Cat. No. 554715), then subsequently stained with 0.25 µg of FITC Mouse Anti-Human IFN-γ antibody (Cat. No. 554700/552887/561053; left panel). To demonstrate specificity of staining, binding by the FITC Mouse Anti-Human IFN-γ antibody was blocked by preincubation of fixed/permeabilized cells with excess Purified Mouse Anti-Human IFN-γ antibody (Cat. No. 554699/550011, right panel). The quadrant markers for the bivariate dot plot were set based on autofluorescence controls, and verified using the unlabeled B27 antibody blocking control.
Product Details
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BD Pharmingen™
IFNG; Interferon-gamma; Interferon-γ; Type II interferon; MAF
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse IgG1, κ
Human IFN-γ Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.5 mg/ml
15978,16176,25712,3458,380793,4049
AB_395517
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The B27 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. The FITC-conjugated B27 antibody (Cat. No. 554700/552887/561053) is especially suitable for these studies. For optimal immunofluorescent staining for flow cytometric analysis, the anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/million cells). For specific methodology, please visit our website, http://www.bdbiosciences.com/us/s/resources and refer to the protocols under "Intracellular Flow".

A useful control for demonstrating specificity of staining is the following: pre-block the paraformaldehyde-fixed/saponin-permeabilized cells with unlabeled B27 antibody (Cat. No. 554699/550011) prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is FITC-MOPC-21 immunoglobulin (Cat. No. 554679); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/million cells).

Neutralization: The NA/LE™ B27 antibody (Cat. No. 554698) is useful for neutralization of human IFN-γ bioactivity. A suitable NA/LE™ mouse IgG1 isotype control to match the NA/LE™ B27 antibody is the 107.3 antibody, Cat. No. 554721.

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. Please note that these applications are not routinely tested BD Biosciences Pharmingen.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. 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.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  6. Cy is a trademark of GE Healthcare.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
554700 Rev. 3
Antibody Details
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B27

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.

554700 Rev. 3
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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FITC
Blue 488 nm
494 nm
518 nm
554700 Rev.3
Citations & References
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Development References (5)

  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. 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: Flow cytometry, Immunoprecipitation, Neutralization). View Reference
  3. 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
  4. 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, Flow cytometry). View Reference
  5. 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
View All (5) View Less
554700 Rev. 3

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