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BV711 Mouse Anti-Human IFN-γ
BV711 Mouse Anti-Human IFN-γ

Two-color flow cytometric analysis of IFN-γ expression in stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated for 6 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 μg/ml final concentration) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), and fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (554722).

       The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD3 antibody (Cat. No. 555335/561810/561811) and either BD Horizon™ BV711 Mouse IgG1 κ Isotype Control (Cat. No. 563044; Left Panel) or BD Horizon BV711 Mouse Anti-Human IFN-γ antibody (Cat. No. 564793; Right Panel). Two-color flow cytometric dot plots showing the correlated expression patterns of IFN-γ (or Ig Isotype control staining) versus CD3 were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Two-color flow cytometric analysis of IFN-γ expression in stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated for 6 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 μg/ml final concentration) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), and fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (554722).

       The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD3 antibody (Cat. No. 555335/561810/561811) and either BD Horizon™ BV711 Mouse IgG1 κ Isotype Control (Cat. No. 563044; Left Panel) or BD Horizon BV711 Mouse Anti-Human IFN-γ antibody (Cat. No. 564793; Right Panel). Two-color flow cytometric dot plots showing the correlated expression patterns of IFN-γ (or Ig Isotype control staining) versus CD3 were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Product Details
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BD Horizon™
IFNG; Interferon-gamma; IFG; IFI; Type II interferon
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse BALB/c IgG1, κ
Human IFN-γ from supernatants of S. aureus-stimulated PBMC
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2738953
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 BD Horizon™ BV711 under optimum conditions, and unconjugated antibody and free BD Horizon™ BV711 were removed.

Recommended Assay Procedures

For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes (including BD Optibuild Brilliant reagents) are used in the same experiment.  Fluorescent dye interactions may cause staining artifacts which may affect data interpretation.  The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions.  More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).

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. 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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  8. Cy is a trademark of GE Healthcare.
  9. BD Horizon Brilliant Violet 711 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,354,239.
  10. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  11. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
564793 Rev. 2
Antibody Details
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4S.B3

The 4S.B3 monoclonal antibody specifically binds to interferon-γ (IFN-γ). The immunogen used to generate this hybridoma was partially purified human IFN-γ obtained from supernatants of human PBMC stimulated with Staphylococcus aureus. Interferon-γ (IFN-γ) is a potent multifunctional cytokine that is 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 antitumor responses of macrophages, NK cells, and neutrophils. In addition, IFN-γ can exert strong regulatory influences on the 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 to direct effects on B cells and T cells themselves. Human IFN-γ is a 14-18 kDa glycoprotein containing 143 amino acid residues.

The antibody was conjugated to BD Horizon BV711 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 711-nm.  BD Horizon BV711 can be excited by the violet laser and detected in a filter used to detect Cy™5.5 / Alexa Fluor® 700-like dyes (eg, 712/20-nm filter).  Due to the excitation and emission characteristics of the acceptor dye, there may be moderate spillover into the Alexa Fluor® 700 and PerCP-Cy5.5 detectors.  However, the spillover can be corrected through compensation as with any other dye combination.

564793 Rev. 2
Format Details
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BV711
The BD Horizon Brilliant Violet™ 711 (BV711) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This tandem fluorochrome is comprised of a BV421 donor with an excitation maximum (Ex Max) of 407-nm and an acceptor dye with an emission maximum (Em Max) at 713-nm. BV711, driven by BD innovation, is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 710-nm (e.g., a 712/20-nm bandpass filter). The acceptor dye can be excited by the Red (628–640-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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BV711
Violet 405 nm
407 nm
713 nm
564793 Rev.2
Citations & References
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Development References (5)

  1. 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. (Clone-specific: Flow cytometry). View Reference
  2. Meager A, Parti S, Barwick S, Spragg J, O'Hagan K. Detection of hybridomas secreting monoclonal antibodies to human gamma interferon using a rapid screening technique and specificity of certain monoclonal antibodies to gamma interferon. J Interferon Res. 1984; 4(4):619-625. (Immunogen: Immunoprecipitation, Radioimmunoassay). View Reference
  3. Meager A. Characterization of interferons and immunoassays. In: Clemens MJ, Morris AG, Gearing AJH, ed. Lymphokines and Interferons. A Practical Approach. Oxford: IRL Press Ltd; 1987:105-127.
  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: 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. (Clone-specific: ELISA). View Reference
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564793 Rev. 2

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