FITC Mouse Anti-Human IFN-γ
Clone B27 (RUO)
- Brand BD Pharmingen™
- Alternative Name IFNG; Interferon-gamma; Interferon-γ; Type II interferon; MAF
- Concentration 0.5 mg/ml
- Isotype Mouse IgG1, κ
- Reactivity Human (QC Testing) Rhesus, Cynomolgus, Baboon (Tested in Development)
Intracellular staining (flow cytometry) (Routinely Tested)
- Immunogen Human IFN-γ Recombinant Protein
- Storage Buffer Aqueous buffered solution containing ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The B27 monoclonal antibody specifically binds to human interferon-ã (IFN-ã), a 14-18 kDa glycoprotein containing 143 amino acid residues. IFN-ã is a potent multifunctional cytokine produced by several activated cell types including NK, NKT, CD4+TCRáâ+, CD8+TCRáâ+, and TCRãä+ T cells. IFN-ã exerts its biological effects through specific binding to the high-affinity IFN-ã receptor complex comprised of IFN-ãRá (CD119) and IFN-ãRâ subunits. In addition to its antiviral effects, IFN-ã 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-ã'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-ã has been described. The B27 antibody has been reported not to bind to denatured IFN-ã.
FITC, fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 Da. FITC is sensitive to pH changes and photobleaching. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. FITC is relatively dim and should be reserved for highly expressed markers whenever possible.
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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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Species testing during development may have been performed with a different format of the same clone. Selected applications have been tested for cross-reactivity.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
Immunofluorescent Staining and Flow Cytometric Analysis: The B27 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. The FITC-conjugated B27 antibody (Cat. No. 554700/552887/561053) is especially suitable for these studies. 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 website, http://www.bdbiosciences.com/us/s/resources and refer to the protocols under "Intracellular Flow".
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. 554699/550011) prior to staining. The staining technique and use of 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-MOPC-21 immunoglobulin (Cat. No. 554679); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/million cells).
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 the 107.3 antibody, Cat. No. 554721.
IP/WB: The 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 these applications are not routinely tested BD Biosciences Pharmingen.