Multicolor flow cytometric analysis of intracellular IFN-γ expression by stimulated mouse splenic CD4-positive and CD4-negative lymphocytes. Mouse BALB/c spleen cells were stimulated (4 hours) with Leukocyte Activation Cocktail with BD GolgiPlug™ (Cat. No. 550583) that contains Phorbol 12-Myristate 13-Acetate (PMA), ionomycin and a protein transport inhibitor, Brefeldin A. The cells were then fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Perm/Wash™ Buffer (Cat No. 554723) and stained with FITC Rat Anti-Mouse CD4 antibody (Cat No. 553055) and either APC-Cy™7 Rat Anti-Mouse IFN-γ antibody (Cat No. 561479, Left Panel) or APC-Cy™7 Rat IgG1, κ Isotype Control (Cat No. 560534, Right Panel). MiCK-1 Mouse Cytokine Positive Control Cells (Cat No. 554652) are prepared in a similar manner. These cells can be used as a positive control for cytokine flow cytometry experiments designed to characterize the nature of mouse IFN-γ-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IFN-γ (or Ig Isotype control staining) versus CD4 were derived from events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Multicolor flow cytometric analysis of intracellular IFN-γ expression by stimulated mouse splenic CD4-positive and CD4-negative lymphocytes. Mouse BALB/c spleen cells were stimulated (4 hours) with Leukocyte Activation Cocktail with BD GolgiPlug™ (Cat. No. 550583) that contains Phorbol 12-Myristate 13-Acetate (PMA), ionomycin and a protein transport inhibitor, Brefeldin A. The cells were then fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Perm/Wash™ Buffer (Cat No. 554723) and stained with FITC Rat Anti-Mouse CD4 antibody (Cat No. 553055) and either APC-Cy™7 Rat Anti-Mouse IFN-γ antibody (Cat No. 561479, Left Panel) or APC-Cy™7 Rat IgG1, κ Isotype Control (Cat No. 560534, Right Panel). MiCK-1 Mouse Cytokine Positive Control Cells (Cat No. 554652) are prepared in a similar manner. These cells can be used as a positive control for cytokine flow cytometry experiments designed to characterize the nature of mouse IFN-γ-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IFN-γ (or Ig Isotype control staining) versus CD4 were derived from events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
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_10898181
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 with APC-Cy7 under optimum conditions, and unconjugated antibody and free APC-Cy7 were removed.
Product Notices
- 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.
- APC-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher.
- 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.
- 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).
- APC-Cy7 is a tandem fluorochrome composed of Allophycocyanin (APC), which is excited by laser lines between 595 and 647 nm and serves as an energy donor, coupled to the cyanine dye Cy7™, which acts as an energy acceptor and fluoresces at 780 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in APC-Cy7, thus maximizing its fluorescence emission intensity, minimizing residual emission from APC, and minimizing required electronic compensation in multilaser-laser flow cytometry systems. Note: Although every effort is made to minimize the lot-to-lot variation in residual emission from APC, it is strongly recommended that every lot be tested for differences in the amount of compensation required and that individual compensation controls are run for each APC-Cy7 conjugate.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
APC-Cy™7 Rat IgG1, κ Isotype Control RUO
Size
0.1 mg
Cat No.
560534
Leukocyte Activation Cocktail, with BD GolgiPlug™ RUO
Size
200 µL
Cat No.
550583
MiCK-1 Mouse Cytokine Positive Control Cells RUO
Size
1 mL
Cat No.
554652
FITC Rat Anti-Mouse CD4 RUO
Size
0.5 mg
Cat No.
553055
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561479 Rev. 2
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.
561479 Rev. 2
Format Details
APC-Cy7
APC-Cy7 dye is a part of the BD APC red family of dyes. This tandem fluorochrome is comprised of a Allophycocyanin (APC) donor that has excitation maxima (Ex Max) of 651 nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 779 nm. APC-Cy7 can be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 780 nm (e.g., a 760/60 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
APC-Cy7
Red 627-640 nm
651 nm
779 nm
561479 Rev.2
Citations & References
View product citations for antibody "561479" on CiteAb
Development References (3)
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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
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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: Flow cytometry). 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. (Methodology: Flow cytometry). View Reference
561479 Rev. 2
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.