Analysis for IFN-γ in 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 ng, Sigma Cat. No. I-0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with PE Rat Anti-Mouse CD4 (Cat. No. 553048) and either the Alexa Fluor® 647 Rat Anti-Mouse IFN-γ antibody (left panel) or with a Alexa Fluor® 647 Rat IgG1 isotype control (Cat. No. 557731), (right panel). To demonstrate specificity of the staining, the binding of the Alexa Fluor® 647 Rat Anti-Mouse IFN-γ antibody (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.
Analysis for IFN-γ in 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 ng, Sigma Cat. No. I-0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with PE Rat Anti-Mouse CD4 (Cat. No. 553048) and either the Alexa Fluor® 647 Rat Anti-Mouse IFN-γ antibody (left panel) or with a Alexa Fluor® 647 Rat IgG1 isotype control (Cat. No. 557731), (right panel). To demonstrate specificity of the staining, the binding of the Alexa Fluor® 647 Rat Anti-Mouse IFN-γ antibody (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
BD Pharmingen™
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_396843
Aqueous buffered solution containing protein stabilizer 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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- 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.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- 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 www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Fixation/Permeabilization Solution Kit with BD GolgiPlug™ RUO
Size
250 Tests
Cat No.
555028
Alexa Fluor® 647 Rat IgG1, κ Isotype Control RUO
Size
0.1 mg
Cat No.
557731
PE Rat Anti-Mouse CD4 RUO
Size
0.1 mg
Cat No.
553048
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Purified Rat Anti-Mouse IFN-γ RUO
Size
0.1 mg
Cat No.
554409
Protein Transport Inhibitor (Containing Brefeldin A) RUO
Size
1 mL
Cat No.
555029
MiCK-1 Mouse Cytokine Positive Control Cells RUO
Size
1 mL
Cat No.
554652
557735 Rev. 3
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.
557735 Rev. 3
Format Details
Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
557735 Rev.3
Citations & References
View product citations for antibody "557735" on CiteAb
Development References (4)
-
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. (Biology). 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. (Biology). View Reference
-
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
-
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. (Biology). View Reference
557735 Rev. 3
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.
Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.