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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 lymphocytes. Human PBMC were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma) and calcium ionophore A23187 (500 ng/ml final concentration; Sigma) in the presence of GolgiStop™ (2 µM final concentration; Cat. No.554724). Stimulated cells were stained with PE Mouse anti-Human CD8 (Cat. No. 555367) and either PE-Cy™7 Mouse anti-Human IFN-γ (Cat. No. 557844/560741/561036, left panel) or PE-Cy™7 mouse IgG1 κ isotype control (Cat. No. 557646, right panel) by using the BD Pharmingen™ staining protocol. 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) and calcium ionophore A23187 (500 ng/ml final concentration; Sigma) in the presence of GolgiStop™ (2 µM final concentration; Cat. No.554724). Stimulated cells were stained with PE Mouse anti-Human CD8 (Cat. No. 555367) and either PE-Cy™7 Mouse anti-Human IFN-γ (Cat. No. 557844/560741/561036, left panel) or PE-Cy™7 mouse IgG1 κ isotype control (Cat. No. 557646, right panel) by using the BD Pharmingen™ staining protocol. 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; 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)
0.2 mg/ml
AB_396894
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 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-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-Cy™7 Mouse IgG1 κ Isotype Control (Cat. No. 557646); use at comparable concentrations to antibody of interest (e.g., ¡ 0.5 µg mAb/1 million cells).

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. 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.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  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. 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).
  8. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  9. Cy is a trademark of GE Healthcare.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
557844 Rev. 6
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.

557844 Rev. 6
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
557844 Rev.6
Citations & References
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Development References (3)

  1. 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. (Biology). View Reference
  2. 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.
  3. 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). View Reference
557844 Rev. 6

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