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 BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit followed by staining with Alexa Fluor® 647 Armenian Hamster anti-Mouse IL-9 (Cat. No. 561463) and PE Rat Anti-Mouse CD4 (Cat. No. 553048). 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. Other compatible fixation and permeabilization treatments are listed in the Suggested Companion Products.
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 BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit followed by staining with Alexa Fluor® 647 Armenian Hamster anti-Mouse IL-9 (Cat. No. 561463) and PE Rat Anti-Mouse CD4 (Cat. No. 553048). 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. Other compatible fixation and permeabilization treatments are listed in the Suggested Companion Products.
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_10644410
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 Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures
This fluorescent antibody is suitable for intracellular staining of mouse leukocytes using BD Cytofix/Cytoperm™ Reagents or BD Phosflow™ Fix Buffer I and Perm/Wash Buffer I (please see Suggested Companion Products).
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
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
Perm/Wash Buffer I RUO
Size
125 mL
Cat No.
557885
561464 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.
561464 Rev. 1
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
561464 Rev.1
Citations & References
Development References (8)
-
Faulkner H, Humphreys N, Renauld JC, Van Snick J, Grencis R. Interleukin-9 is involved in host protective immunity to intestinal nematode infection. Eur J Immunol. 1997; 27(10):2536-2540. (Biology). View Reference
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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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Petit-Frere C, Dugas B, Braquet P, Mencia-Huerta JM. Interleukin-9 potentiates the interleukin-4-induced IgE and IgG1 release from murine B lymphocytes. Immunology. 1993; 79(1):146-151. (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
561464 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.
