RB780 Mouse Anti-Human IFN-γ

BD Horizon™ RB780 Mouse Anti-Human IFN-γ

Name: RB780 Mouse Anti-Human IFN-γ
Brand: BD Horizon™
SKU: 568687

Clone B27

(RUO)

Flow cytometric analysis of IFN-γ expression in stimulated Human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; 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 with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either BD Horizon™ RB780 Mouse IgG1, κ Isotype Control (Cat No. 568532; Left Plot) or BD Horizon™ RB780 Mouse Anti-Human IFN-γ antibody (Cat No.568686/568687; Right Plot). The two-parameter pseudocolor density plot showing the correlated expression of IFN-γ (or Ig Isotype control staining) versus side light-scatter (SSC-A) signals was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Flow Cytometer System and FlowJo™ software.

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Product Details

Brand: BD Horizon™

Alternative Name: IFNG; Interferon-gamma; Interferon-γ; Type II interferon; MAF

Reactivity: Human (QC Testing), Rhesus,Cynomolgus,Baboon (Tested in Development)

Isotype: Mouse IgG1, κ

Immunogen: Human IFN-γ Recombinant Protein

Application: Intracellular staining (flow cytometry) (Routinely Tested)

Vol. Per Test: 5 µl

Entrez Gene ID: 3458

Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.

Regulatory Status: 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 to the dye under optimum conditions and unreacted dye was removed.

Recommended Assay Procedures

BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.

Product Notices

Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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).
When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
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.
Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
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.

Companion Products

Protein Transport Inhibitor (Containing Monensin) RUO

Size 0.7 mL Cat No. 554724

Perm/Wash Buffer RUO

Size 100 mL Cat No. 554723

Fixation Buffer RUO

Size 100 mL Cat No. 554655

568687 Rev. 2

Antibody Details

B27

The B27 monoclonal antibody specifically binds to human interferon-γ (IFN-γ), a 14-18 kDa glycoprotein containing 143 amino acid residues. IFN-γ is a potent multifunctional cytokine 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 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-γ'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-γ has been described. The B27 antibody has been reported not to bind to denatured IFN-γ.

568687 Rev. 2

Format Details

RB780

The BD Horizon RealBlue™ 780 (RB780) Dye is part of the BD family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 781-nm. Driven by BD innovation, RB780 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB780 can be used as an alternative to PE-Cy7 and we recommend using an optical filter centered near 780-nm (eg, a 780/60-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-Cy7. RB780 is on average brighter than PE-Cy7 and has minimal spillover into Yellow-Green detectors.

Format: RB780

Excitation Source: Blue 488 nm

Excitation Max: 498 nm

Emission Max: 781 nm

568687 Rev.2

Citations & References

Development References (8)

Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific: ELISA, Neutralization). View Reference
Draghi M, Yawata N, Gleimer M, Yawata M, Valiante NM, Parham P. Single-cell analysis of the human NK cell response to missing self and its inhibition by HLA class I.. Blood. 2005; 105(5):2028-35. (Clone-specific). View Reference
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. (Clone-specific: ELISA, Immunoprecipitation, Neutralization). View Reference
Pitcher CJ, Hagen SI, Walker JM, et al. Development and homeostasis of T cell memory in rhesus macaque. J Immunol. 2002; 168(1):29-43. (Clone-specific: Flow cytometry). View Reference
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, IC/FCM Block). View Reference
Rosato PC, Wijeyesinghe S, Stolley JM, et al. Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy.. Nat Commun. 2019; 10(1):567. (Clone-specific: Flow cytometry). View Reference
Satooka H, Ishigaki H, Todo K, et al. Characterization of tumour-infiltrating lymphocytes in a tumour rejection cynomolgus macaque model. Scientific reports. 2020; 10(1):8414. (Clone-specific: Flow cytometry). View Reference
Trotta E, Bessette PH, Silveria SL, et al. A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism.. Nat Med. 2018; 24(7):1005-1014. (Clone-specific: Flow cytometry). View Reference

View All (8)

568687 Rev. 2

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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.