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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 hour with hamster anti-mouse CD3 (2 µg/ml, 145-2C11, Cat. No. 553057) and hamster anti-mouse CD28 (2 µg/ml, 37.51, Cat. No, 553294) antibodies in the presence of 2 µM GolgiStop™ (aka, monensin; Cat. No. 554724). The splenocytes were harvested, stained with 0.06 µg of FITC rat anti-mouse CD8 (FITC 53-6.7, Cat. No. 553030), fixed, permeabilized, and subsequently stained with 0.06 µg of PE rat anti-mouse IFN-γ (PE-XMG1.2, Cat. No. 554412, left panel) by using Pharmingen's staining protocol. 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; see 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™ PE 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 XMG1.2 antibody is useful for the immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. The PE-conjugated XMG1.2 antibody is especially suitable for these studies. For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/million cells). A useful control for demonstrating specificity of staining is either of the following: (1) pre-block the fluorochrome-conjugated XMG1.2 antibody prior to staining, with unlabeled XMG1.2 antibody (Cat. No. 554409), or (2) pre-block with a molar excess of ligand, (e.g. recombinant mouse IFN-g; Cat. No. 554587) prior to staining.
A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse or human cells is PE-R3-34 immunoglobulin (Cat. No. 554685); use at comparable concentrations to antibody of interest.
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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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, Neutralization). 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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For Research Use Only. Not for use in diagnostic or therapeutic procedures.