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Alexa Fluor® 647 Mouse Anti-Human IFN-γ
Alexa Fluor® 647 Mouse Anti-Human IFN-γ

Expression of IFN-γ by stimulated human peripheral blood lymphocytes. 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) and stained with PE-Cy™7 Mouse Anti-Human CD8 (Cat. No. 557746/557750/560917) and either Alexa Fluor® 647 Mouse Anti-Human IFN-γ (Cat. No. 557729; left panel) or Alexa Fluor® 647 Mouse IgG1 κ Isotype Control (Cat. No. 557732; right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor® 647 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. Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes. 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 lymphocytes. 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) and stained with PE-Cy™7 Mouse Anti-Human CD8 (Cat. No. 557746/557750/560917) and either Alexa Fluor® 647 Mouse Anti-Human IFN-γ (Cat. No. 557729; left panel) or Alexa Fluor® 647 Mouse IgG1 κ Isotype Control (Cat. No. 557732; right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor® 647 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. Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes. 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_396837
Aqueous buffered solution containing BSA and ≤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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The FITC-, PE-, APC-, PE-Cy7-, Alexa Fluor® 488-, and Alexa Fluor® 647-conjugated B27 antibodies (Cat. No. 554700, 554701, 554702, 557643, 557718, and 557729) are  useful for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. For optimal immunofluorescent staining this antibody should be used at 5 µl per test. 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. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is Alexa Fluor® 647-MOPC-21 (Cat. No. 557732); use at 5 µl/test.

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 107.3 antibody (Cat. No. 554721).

IP/WB: The unconjugated 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 this application is not routinely tested at BD Biosciences Pharmingen.

Product Notices

  1. 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).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
557729 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.

557729 Rev. 3
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
557729 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: Immunoprecipitation, Western blot). 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). 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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557729 Rev. 3

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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.