Two-color flow cytometric analysis of IFN-γ expression in stimulated Mouse splenocytes. BALB/c Mouse splenic leucocytes 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 APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and either BD Horizon™ RY586 Rat IgG1, κ Isotype Control (Cat. No. 568822; Left Plot) or BD Horizon™ RY586 Rat Anti-Mouse IFN-γ antibody (Cat. No. 568549/568550; Right Plot) at 0.25 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. Bivariate pseudocolor density plots showing the correlated expression of IFN-γ (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated leucocytes. 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 splenocytes. BALB/c Mouse splenic leucocytes 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 APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and either BD Horizon™ RY586 Rat IgG1, κ Isotype Control (Cat. No. 568822; Left Plot) or BD Horizon™ RY586 Rat Anti-Mouse IFN-γ antibody (Cat. No. 568549/568550; Right Plot) at 0.25 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. Bivariate pseudocolor density plots showing the correlated expression of IFN-γ (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated leucocytes. 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
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 to the dye under optimum conditions and unreacted dye was removed.
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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- CF™ is a trademark of Biotium, Inc.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
RY586 Rat IgG1, κ Isotype Control RUO
Size
0.1 mg
Cat No.
568822
568549 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.
568549 Rev. 1
Format Details
RY586
The BD Horizon RealYellow™ 586 (RY586) Dye is part of the BD family of yellow-green dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 565-nm and an emission maximum (Em Max) at 586-nm. Driven by BD innovation, RY586 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), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE. Compared to PE, RY586 is similar in brightness, minimal spillover into Blue detectors, and increased spillover into the 610/20-nm (PE-CF594) detector. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
RY586
Yellow-Green 561 nm
564 nm
586 nm
568549 Rev.1
Citations & References
View product citations for antibody "568549" 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
568549 Rev. 1
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.