Multicolor flow cytometric analysis of IL-9 expression by unstimulated and activated mouse spleen cells. Mouse spleen cells were either unstimulated (Left Panel) or stimulated in a tissue culture plate coated with Anti-Mouse CD3e and soluble Anti-Mouse CD28 antibodies along with Recombinant Mouse IL-2, IL-4, and TGF-β proteins and Anti-Mouse IFN-γ antibody for 4 days. On day 4 the cells were harvested and restimulated with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139) plus Ionomycin (Sigma; I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor for 5 hours (Right Panel). The cells were then fixed and permeabilized using a BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit followed by staining with PerCP-Cy™5.5 Armenian Hamster Anti-Mouse IL-9 (Cat. No. 561492) and Alexa Fluor® 700 Rat Anti-Mouse CD4 (Cat. No. 561025/557956). Two-color flow cytometric dot plots showing the correlated expression patterns of CD4 versus IL-9 were derived from gated 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 IL-9 expression by unstimulated and activated mouse spleen cells. Mouse spleen cells were either unstimulated (Left Panel) or stimulated in a tissue culture plate coated with Anti-Mouse CD3e and soluble Anti-Mouse CD28 antibodies along with Recombinant Mouse IL-2, IL-4, and TGF-β proteins and Anti-Mouse IFN-γ antibody for 4 days. On day 4 the cells were harvested and restimulated with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139) plus Ionomycin (Sigma; I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor for 5 hours (Right Panel). The cells were then fixed and permeabilized using a BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit followed by staining with PerCP-Cy™5.5 Armenian Hamster Anti-Mouse IL-9 (Cat. No. 561492) and Alexa Fluor® 700 Rat Anti-Mouse CD4 (Cat. No. 561025/557956). Two-color flow cytometric dot plots showing the correlated expression patterns of CD4 versus IL-9 were derived from gated 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™
IL-9; Interleukin-9; MEA; P40; T-cell growth factor P40; TCGF III
Mouse (QC Testing)
Armenian Hamster IgG2, κ
Mouse IL-9 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_10697048
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 with PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- 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.
- PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
- PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
- This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
- 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 www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Fixation/Permeabilization Kit RUO
Size
250 Tests
Cat No.
554714
Fixation and Permeabilization Solution RUO
Size
125 mL
Cat No.
554722
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Fix Buffer I RUO
Size
250 mL
Cat No.
557870
561492 Rev. 1
Antibody Details
D9302C12
The D9302C12 monoclonal antibody specifically binds to the multifunctional mouse cytokine, Interleukin-9 (IL-9). IL-9 is a 126 amino acid-long glycoprotein that is produced by various subsets of activated CD4+ T cells. IL-9 acts on target cells by binding to and signaling through the heterodimeric IL-9 receptor (IL-9R) complex that is comprised of transmembrane IL-9 receptor alpha (IL-9Rα) and common gamma chain (γc) subunits. IL-9 can promote the survival, growth, proliferation and/or differentiation of various cell types including thymocytes, T cells, B cells, mast cells, and hematopoietic progenitor cells. IL-9 can augment IL-4-induced IgE and IgG1 production from lipopolysaccharide-primed mouse B cells and induce granzyme and high-affinity IgE receptor gene expression by mouse T helper cell clones and mast cell lines. IL-9 plays an important role in vivo in helminth elimination. The D9302C12 antibody neutralizes mouse IL-9 bioactivity.
561492 Rev. 1
Format Details
PerCP-Cy5.5
PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-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.
PerCP-Cy5.5
Blue 488 nm
482 nm
676 nm
561492 Rev.1
Citations & References
Development References (6)
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Hultner L, Druez C, Moeller J, et al. Mast cell growth-enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin 9). Eur J Immunol. 1990; 20(6):1413-1416. (Biology). View Reference
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Louahed J, Kermouni A, Van Snick J, Renauld JC. IL-9 induces expression of granzymes and high-affinity IgE receptor in murine T helper clones. J Immunol. 1995; 154(10):5061-5070. (Biology). View Reference
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Renauld JC, Kermouni A, Vink A, Louahed J, Van Snick J. Interleukin-9 and its receptor: involvement in mast cell differentiation and T cell oncogenesis. J Leukoc Biol. 1995; 57(3):353-360. (Biology). View Reference
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Suda T, Murray R, Fischer M, Yokota T, Zlotnik A. Tumor necrosis factor-alpha and P40 induce day 15 murine fetal thymocyte proliferation in combination with IL-2. J Immunol. 1990; 144(5):1783-1787. (Biology). View Reference
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Van Snick J, Cayphas S, Vink A, et al. Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci U S A. 1986; 83(24):9679-9683. (Biology). View Reference
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Van Snick J, Goethals A, Renauld JC, et al. Cloning and characterization of a cDNA for a new mouse T cell growth factor (P40). J Exp Med. 1989; 169(1):363-368. (Biology). View Reference
561492 Rev. 1
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.
