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PE-Cy™7 Mouse Anti-Human IFN-γ
PE-Cy™7 Mouse Anti-Human IFN-γ

Flow cytometric analysis for IFN-γ in stimulated Rhesus macaque peripheral blood mononuclear cells (PBMC).  PBMC from Rhesus macaque were stimulated for 6 hours with 50 ng/mL PMA (Sigma-Aldrich Cat. No. P-8139) and 500 ng/mL calcium ionophore A23187 (Sigma-Aldrich Cat. No. C-9275) in the presence of BD GolgiStop™ (Cat. No. 554724).  Cells were then fixed and permeabilized using BD Cytofix/Cytoperm™ (Cat. No. 554714) followed by staining with either a PE-Cy™7 Mouse IgG1, κ isotype control (Cat. No. 557646; left panel) or with the PE-Cy™7 Mouse Anti-Human IFN-γ antibody (Cat. No. 557844/560472/557844; right panel).  Dot plots were derived from gated events based on light scattering characteristics for lymphocytes.  Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Flow cytometric analysis for IFN-γ in stimulated Rhesus macaque peripheral blood mononuclear cells (PBMC).  PBMC from Rhesus macaque were stimulated for 6 hours with 50 ng/mL PMA (Sigma-Aldrich Cat. No. P-8139) and 500 ng/mL calcium ionophore A23187 (Sigma-Aldrich Cat. No. C-9275) in the presence of BD GolgiStop™ (Cat. No. 554724).  Cells were then fixed and permeabilized using BD Cytofix/Cytoperm™ (Cat. No. 554714) followed by staining with either a PE-Cy™7 Mouse IgG1, κ isotype control (Cat. No. 557646; left panel) or with the PE-Cy™7 Mouse Anti-Human IFN-γ antibody (Cat. No. 557844/560472/557844; right panel).  Dot plots were derived from gated events based on light scattering characteristics for lymphocytes.  Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Product Details
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BD Pharmingen™
IFNG; Interferon-gamma; IFG; IFI; Type II interferon
Rhesus, Cynomolgus, Baboon (QC Testing), Human (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_1727530
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

        Flow Cytometric Analysis: The PE-Cy™7 Mouse Anti-Human IFN-γ antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. For specific methodology, please visit our web site, http://www.bdbiosciences.com/us/s/resources, and go to the protocols section under "Intracellular Staining".

        A useful control for demonstrating specificity of staining is pre-block the fixed/permeabilized cells with Purified Mouse Anti-Human IFN-γ (Cat. No. 554549) prior to staining. The 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 PE-Cy7 Mouse IgG1¦Isotype Control (Cat. No. 557646); use at comparable concentrations to antibody of interest (e.g., ¡ 0.5 ¦g mAb/1 million cells).

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. 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).
  6. 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.
  7. PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  10. Cy is a trademark of GE Healthcare.
  11. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  12. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560741 Rev. 4
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.

Clone 4S.B3 also cross-reacts with a cytoplasmic component of peripheral blood CD3+ lymphocytes of baboon, and both rhesus and cynomolgus macaque monkeys following five-hour treatment with phorbol myristic acetate (PMA) and Ca++ ionophore (A23187) in the presence of monensin. The staining pattern of 4S.B3 in CD3+ cells is similar to that observed with peripheral blood T lymphocytes from normal human donors. This reagent is useful for intracellular immunofluorescent staining for flow cytometric analysis to identify and enumerate IFN-γ + cells within a mixed cell population.

560741 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
560741 Rev.4
Citations & References
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Development References (6)

  1. Farrar MA, Schreiber RD. The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol. 1993; 11:571-611. (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. (Biology). 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. Sester U, Sester M, Hauk M, Kaul H, Köhler H, Girndt M. T-cell activation follows Th1 rather than Th2 pattern in haemodialysis patients.. Nephrol Dial Transplant. 2000; 15(8):1217-1223. (Biology). View Reference
  6. Vogel S, Friedman R, Hogan M. Measurement of antiviral activity induced by interferons a, b, and g.. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, ed. Current Protocols in Immunology. New York: John Wiley & Sons; 2007:6.9.1-6.9.15.
View All (6) View Less
560741 Rev. 4

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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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.