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PE-Cy™7 Mouse Anti-Human IFN-γ
PE-Cy™7 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). Cells were harvested, fixed and permeabilized with BD Cytofix/Cytoperm Plus Kit (with BD GolgiStop) (Cat. No. 554715), then stained with PE Mouse Anti-Human CD8  (Cat. No. 555367/561950/561949/557086) and either PE-Cy™7 Mouse Anti-Human IFN-γ (Cat. No. 557643/560924, left panel) or PE-Cy™7 Mouse IgG1 κ Isotype Control (Cat. No. 557646, right panel) as a specificity control. To demonstrate additional specificity of staining the binding of PE-Cy™7 Mouse Anti-Human IFN-γ was blocked by preincubation of the fixed/permeabilized cells with an excess of Purified Mouse Anti-Human IFN-γ (5 µg, Cat. No. 554699/550011, data not shown) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.

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). Cells were harvested, fixed and permeabilized with BD Cytofix/Cytoperm Plus Kit (with BD GolgiStop) (Cat. No. 554715), then stained with PE Mouse Anti-Human CD8  (Cat. No. 555367/561950/561949/557086) and either PE-Cy™7 Mouse Anti-Human IFN-γ (Cat. No. 557643/560924, left panel) or PE-Cy™7 Mouse IgG1 κ Isotype Control (Cat. No. 557646, right panel) as a specificity control. To demonstrate additional specificity of staining the binding of PE-Cy™7 Mouse Anti-Human IFN-γ was blocked by preincubation of the fixed/permeabilized cells with an excess of Purified Mouse Anti-Human IFN-γ (5 µg, Cat. No. 554699/550011, data not shown) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.

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)
5 µl
AB_396760
Aqueous buffered solution containing BSA and ≤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 PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The PE-Cy7-conjugated B27 antibody (Cat. No. 557643) is useful for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. 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) prior to staining. The intracellular cytokine staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit the protocols section under "Intracellular Flow" on our web site,

http://www.bdbiosciences.com/us/s/resources.

Neutralization: The NA/LE B27 antibody (Cat. No. 554698) is useful for neutralization of human IFN-γ bioactivity.

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 at BD Biosciences Pharmingen.

Product Notices

  1. An isotype control should be used at the same concentration as the antibody of interest.
  2. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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.
  5. APC-Cy7 is a tandem fluorochrome composed of Allophycocyanin (APC), which is excited by laser lines between 595 and 647 nm and serves as an energy donor, coupled to the cyanine dye Cy7™, which acts as an energy acceptor and fluoresces at 780 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in APC-Cy7, thus maximizing its fluorescence emission intensity, minimizing residual emission from APC, and minimizing required electronic compensation in multilaser-laser flow cytometry systems. Note: Although every effort is made to minimize the lot-to-lot variation in residual emission from APC, it is strongly recommended that every lot be tested for differences in the amount of compensation required and that individual compensation controls are run for each APC-Cy7 conjugate.
  6. APC-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher.
  7. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
  10. Cy is a trademark of GE Healthcare.
  11. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  12. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
557643 Rev. 4
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.

557643 Rev. 4
Format Details
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PE-Cy7
PE-Cy7 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 781-nm. PE can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 781 nm (e.g., a 760/60-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the Red (627–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE-Cy7
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
781 nm
557643 Rev.4
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. (Biology). 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: 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. (Biology: 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
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557643 Rev. 4

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