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Expression of IFN-γ by stimulated CD8+ and CD8-BALB/c spleen cells. Splenocytes from 6 month old BALB/c mice were cultured for 3 days in the presence of SEB (2 µg/ml; Sigma, Cat. No. S-4881), then restimulated for 5 hr with hamster anti-mouse CD3 (2 µg/ml, 145-2C11, Cat. #553057) and hamster anti-mouse CD28 (2 µg/ml, 37.51, Cat. No. 553294) antibodies in the presence of 2 µg GolgiStop™ (aka, Monensin; Cat. No. 554724). The splenocytes were harvested, stained with 0.06 µg of FITC-conjugated rat anti-mouse CD48 (FITC-53-6.7 Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.06 µg of PE-conjugated rat anti-mouse IFN-γ antibody (PE-XMG1.2) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-XMG1.2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled XMG1.2 antibody (5.0 µg; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
BD Pharmingen™ Purified Rat Anti-Mouse IFN-γ
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
Immunofluorescent Staining and Flow Cytometric Analysis: The purified XMG1.2 antibody can be used as a blocking control to demonstrate specificity of IFN-γ staining by directly conjugated-XMG1.2 (PE: Cat. No. 554412; FITC: Cat. No. 554411; APC: Cat. No. 554413; PE-Cy7: Cat. No. 557649). To perform this control, the fixed/permeabilized cells (~1 million) can be incubated with 1-10 µg of unlabeled XMG1.2 antibody (Cat. No. 554409) for 20 minutes at 4°C, prior to staining with conjugated-XGM1.2 antibody.
Western blot: The purified XMG1.2 antibody (Cat. No. 554409) has been reported to be useful for Western blotting. Please note that this application is not routinely tested at BD Biosciences Pharmingen.
ELISA Detection: The biotinylated XMG1.2 antibody (Cat. No. 554410) is useful as a detection antibody for a sandwich ELISA for measuring mouse IFN-γ protein levels and can be used in combination with purified R4-6A2 (Cat. No. 551216) as the capture antibody and recombinant mouse IFN-γ as the standard. This ELISA pair is recommended primarily for measuring cytokine from experimental cell culture systems. These ELISA reagents are not recommended for assaying serum or plasma samples. For measuring Mouse IFN-γ in serum or plasma our Mouse IFN-γ BD OptEIA™ Set (Cat. No. 551866) or BD OptEIA Kit (Cat. No. 558258) are specially formulated and recommended.
Neutralization/Blocking: The NA/LE™ format of the XMG1.2 antibody (Cat. No. 554408) is useful for neutralization of mouse IFN-γ bioactivity.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
Companion Products
The XMG1.2 monoclonal antibody specifically binds to mouse interferon-γ (IFN-γ) protein. IFN-γ is a pleiotropic cytokine, of approximately 15-17 kDa, involved in the regulation of inflammatory and immune responses. It plays an important role in activation, growth, and differentiation of T and B lymphocytes, macrophages, NK cells and other non-hematopoietic cell types. IFN-γ production is associated with the Th1 cell differentiation. The purified form of this antibody has been reported to be a neutralizing antibody.
Development References (4)
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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). View Reference
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Cherwinski HM, Schumacher JH, Brown KD, Mosmann TR. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J Exp Med. 1987; 166(5):1229-1244. (Clone-specific). View Reference
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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: Blocking, Flow cytometry). View Reference
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Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Clone-specific). View Reference
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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.