Two-color flow cytometric analysis of IFN-γ expression in stimulated Mouse splenic leukocytes. BALB/c Mouse splenocytes were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139; 50 ng/ml) and Ionomycin (Sigma I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) [Cat. No. 554724]. The cells were harvested and 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 FITC Rat Anti-Mouse CD4 antibody (Cat. No. 553047) and with either BD Horizon™ RY610 Rat IgG1, κ Isotype Control (Cat. No. 571676; Left Plot) or BD Horizon™ RY610 Rat Anti-Mouse IFN-γ antibody (Cat. No. 571357/571359; Right Plot) at 0.06 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. The bivariate pseudocolor density plot showing the correlated expression of IFN-γ (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact stimulated leukocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Two-color flow cytometric analysis of IFN-γ expression in stimulated Mouse splenic leukocytes. BALB/c Mouse splenocytes were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139; 50 ng/ml) and Ionomycin (Sigma I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) [Cat. No. 554724]. The cells were harvested and 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 FITC Rat Anti-Mouse CD4 antibody (Cat. No. 553047) and with either BD Horizon™ RY610 Rat IgG1, κ Isotype Control (Cat. No. 571676; Left Plot) or BD Horizon™ RY610 Rat Anti-Mouse IFN-γ antibody (Cat. No. 571357/571359; Right Plot) at 0.06 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. The bivariate pseudocolor density plot showing the correlated expression of IFN-γ (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact stimulated leukocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Product Details
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
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 to the dye under optimum conditions and unreacted dye was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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.
- 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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
- CF™ is a trademark of Biotium, Inc.
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
RY610 Rat IgG1, κ Isotype Control RUO
Size
0.1 mg
Cat No.
571676
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
571359 Rev. 1
Antibody Details
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.
571359 Rev. 1
Format Details
RY610
The BD Horizon RealYellow™ 610 (RY610) Dye is part of the BD® family of yellow-green dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 557-nm and an emission maximum (Em Max) at 610-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RY610 can be used on both spectral and conventional cytometers and is designed to be excited by the Yellow-Green laser (561-nm) with minimal excitation by the 488-nm blue laser. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY610 can be used as an alternative to PE-CF594 and we recommend using an optical filter centered near 610-nm (eg, a 610/20-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE-CF594.
RY610
Yellow-Green 561 nm
557 nm
610 nm
571359 Rev.1
Citations & References
View product citations for antibody "571359" on CiteAb
Development References (7)
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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: ELISA, Neutralization). 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: ELISA). View Reference
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Klinman D and Nutman T. ELISPOT assay to detect cytokine-secreting murine and human cells. In: Coligan J, Kruisbeek A, Margulies D, Shevach E, Strober W, ed. Current Protocols in Immunology. 1994:6-19.
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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). 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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Suzuki Y, Yang Q, Conley FK, Abrams JS, Remington JS. Antibody against interleukin-6 reduces inflammation and numbers of cysts in brains of mice with toxoplasmic encephalitis. Infect Immun. 1994; 62(7):2773-2778. (Clone-specific: In vivo exacerbation, Neutralization). View Reference
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Yang X, HayGlass KT. A simple, sensitive, dual mAb based ELISA for murine gamma interferon determination: comparison with two common bioassays. J Immunoassay. 1993; 14(3):129-148. (Clone-specific: ELISA). View Reference
571359 Rev. 1
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.