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PE-CF594 Rat Anti-Mouse IFN-γ
PE-CF594 Rat Anti-Mouse IFN-γ
Multicolor flow cytometric analysis of IFN-γ expressed in MiCK-1 cells. Mouse intracellular CytoKine-1 (MiCK-1) Positive Control Cells (Cat. No. 554652) were washed and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained with APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051) and either BD Horizon™ PE-CF594 Rat IgG1, κ Isotype Control (Cat. No. 562309; Left Panel) or BD Horizon™ PE-CF594 Rat Anti-Mouse IFN-γ antibody (Cat. No. 562303 / 562333; Right Panel). The two-color flow cytometric dot plots showing IFN-γ (or Ig Isotype Control staining) versus CD4 were derived from events with the forward and side light-scatter characteristics of intact cells. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Multicolor flow cytometric analysis of IFN-γ expressed in MiCK-1 cells. Mouse intracellular CytoKine-1 (MiCK-1) Positive Control Cells (Cat. No. 554652) were washed and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained with APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051) and either BD Horizon™ PE-CF594 Rat IgG1, κ Isotype Control (Cat. No. 562309; Left Panel) or BD Horizon™ PE-CF594 Rat Anti-Mouse IFN-γ antibody (Cat. No. 562303 / 562333; Right Panel). The two-color flow cytometric dot plots showing IFN-γ (or Ig Isotype Control staining) versus CD4 were derived from events with the forward and side light-scatter characteristics of intact cells. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Product Details
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BD Horizon™
Ifg; Ifng; IFN-γ; IFN-g; IFN-gamma; Interferon gamma; Type II Interferon
Mouse (QC Testing)
Rat IgG1, κ
Mouse IFN-γ Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
15978
AB_11153140
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
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 with BD Horizon™ PE-CF594 under optimum conditions, and unconjugated antibody and free PE-CF594 were removed.

Recommended Assay Procedures

Flow cytometry:  The XMG1.2 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations.  A useful control investigators may consider using for demonstrating specificity of staining, is to pre-block with one of the following reagents: (1) recombinant mouse IFN-γ (Cat. No. 554587) or (2) unlabeled XMG1.2 antibody (Cat. No. 554409), prior to staining.

Cell Preparation: Investigators not wishing to utilize MiCK-1 cells may alternatively prepare mouse splenocytes (e.g BALB/c) stimulated for 4-6 hours with PMA (5 ng/mL, Sigma-Aldrich Cat. No. P-8139) and ionomycin (500 ng, Sigma-Aldrich Cat. No. I-0634) in the presence of 1 µg/mL Brefeldin A (BD GolgiPlug™ MN 555029).  Investigators are advised to fix and permeabilize the cells prior to staining.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. When excited by the yellow-green (561-nm) laser, the fluorescence may be brighter than when excited by the blue (488-nm) laser.
  3. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using multi-laser cytometers, which may directly excite both PE and CF™594.
  4. An isotype control should be used at the same concentration as the antibody of interest.
  5. 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.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. CF™ is a trademark of Biotium, Inc.
  8. This product is provided under an Agreement between BIOTIUM and BD Biosciences. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications owned or licensed by Biotium, Inc. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
  9. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  10. 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.
  11. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
562333 Rev. 3
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.

This antibody is conjugated to BD Horizon™ PE-CF594, which has been developed exclusively by BD Biosciences as a better alternative to PE-Texas Red. PE-CF594 excites and emits at similar wavelengths to PE-Texas Red yet exhibits improved brightness and spectral characteristics. Due to PE having maximal absorption peaks at 496 nm and 564 nm, PE-CF594 can be excited by the blue (488-nm), green (532-nm) and yellow-green (561-nm) lasers and can be detected with the same filter set as PE-Texas Red (eg 610/20-nm filter).

562333 Rev. 3
Format Details
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PE-CF594
BD Horizon™ PE-CF594 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 with an emission maximum (Em Max) at 615-nm. PE-CF594, driven by BD innovation, is designed to be excited by the blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 615 nm (e.g., a 610/20-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 green (532-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-CF594
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
615 nm
562333 Rev.3
Citations & References
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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: Blocking, Neutralization). 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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562333 Rev. 3

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.