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PE-Cy™7 Rat Anti-Mouse IFN-γ
PE-Cy™7 Rat Anti-Mouse IFN-γ
Expression of IFN- by stimulated CD4+ and CD4-BALB/c spleen cells. Splenocytes from BALB/C mice were stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and Ionomycin (500 µg/ml, Sigma Cat. No. I-0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with APC rat anti-mouse CD4 (APC-RM4-5, Cat. No. 553051) and either rat anti-mouse IFN-γ antibody (PE-Cy7-XMG1.2, Cat. No. 557649), (left panel) or immunoglobulin isotype control (PE-Cy7-R3-34, Cat. No. 557645), (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of PE-Cy7-XMG1.2 was blocked by preincubation of the fixed/permeabilized cells with an excess of unlabelled XMG1.2 antibody (5 µg, Cat. No. 554409, data not shown) prior to stainining. The quadarant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IFN- by stimulated CD4+ and CD4-BALB/c spleen cells. Splenocytes from BALB/C mice were stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and Ionomycin (500 µg/ml, Sigma Cat. No. I-0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with APC rat anti-mouse CD4 (APC-RM4-5, Cat. No. 553051) and either rat anti-mouse IFN-γ antibody (PE-Cy7-XMG1.2, Cat. No. 557649), (left panel) or immunoglobulin isotype control (PE-Cy7-R3-34, Cat. No. 557645), (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of PE-Cy7-XMG1.2 was blocked by preincubation of the fixed/permeabilized cells with an excess of unlabelled XMG1.2 antibody (5 µg, Cat. No. 554409, data not shown) prior to stainining. The quadarant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Product Details
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG1, κ
Mouse IFN-γ Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
15978
AB_396766
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The PE-Cy-7 conjugated 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. For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.125 µg mAb/million cells).  A useful control for demonstrating specificity of staining is to pre-block the fixed/permeabilized cells with unlabeled XMG1.2 antibody (Cat. No. 554409) 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. 557645); use at comparable concentrations to antibody of interest.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
  4. 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.
  5. PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. 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.
  8. Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
  9. This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
557649 Rev. 4
Antibody Details
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XMG1.2

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.

557649 Rev. 4
Format Details
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PE-Cy7
PE-Cy7 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 781-nm. PE can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 781 nm (e.g., a 760/60-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the Red (627–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE-Cy7
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
781 nm
557649 Rev.4
Citations & References
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View product citations for antibody "557649" on CiteAb

Development References (4)

  1. 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
  2. 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
  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: Flow cytometry). View Reference
  4. 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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557649 Rev. 4

 

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