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FITC Mouse Anti-Human IFN-γ
FITC 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) and calcium ionophore A23187 (500 ng/ml final concentration; Sigma) in the presence of GolgiStop™ (2 µM final concentration; Cat. No.554724). The PBMC were stained with PE-Cy™5 Mouse Anti-Human CD3 (Cat.No. 555334/561007), fixed, permeabilized, and subsequently stained with 0.25 µg of FITC Mouse Anti-Human IFN-γ (Cat. No.555334/561057/552882) by using Pharmingen's staining protocol (left panel). The binding of FITC Mouse Anti-Human IFN-γ was blocked by preincubation of cells with Purified Mouse Anti-Human IFN-γ (5 µg; Cat. No. 554549; right panel). The quadrant markers for the bivariate dot plot were set based on the autofluorescence controls and verified using the unlabeled antibody and ligand blocking 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) and calcium ionophore A23187 (500 ng/ml final concentration; Sigma) in the presence of GolgiStop™ (2 µM final concentration; Cat. No.554724). The PBMC were stained with PE-Cy™5 Mouse Anti-Human CD3 (Cat.No. 555334/561007), fixed, permeabilized, and subsequently stained with 0.25 µg of FITC Mouse Anti-Human IFN-γ (Cat. No.555334/561057/552882) by using Pharmingen's staining protocol (left panel). The binding of FITC Mouse Anti-Human IFN-γ was blocked by preincubation of cells with Purified Mouse Anti-Human IFN-γ (5 µg; Cat. No. 554549; right panel). The quadrant markers for the bivariate dot plot were set based on the autofluorescence controls and verified using the unlabeled antibody and ligand blocking 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)
0.5 mg/ml
AB_395473
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 FITC under optimum conditions, and unreacted FITC was removed.

Recommended Assay Procedures

Immunofluorescent Staining for Flow Cytometric Analysis: The FITC 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 optimal immunofluorescent staining for flow cytometric analysis, the anti-cytokine antibody should be titrated (¡ 0.5 µg mAb/million cells). 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 FITC Mouse IgG1, κ Isotype Control (Cat. No. 554679); 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. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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.

Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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FITC
Blue 488 nm
494 nm
518 nm
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

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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.