Flow cytometric analysis of IL-9 in stimulated human CD4-positive T cells. Human peripheral blood mononuclear cells were stimulated in a tissue culture plate coated with NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 µg/ml, coated overnight at 4°C) and soluble NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 1 µg/ml) antibodies plus recombinant Human IL-2 (Cat. No. 554603; 10 ng/ml), IL-4 (Cat. No. 554605; 50 ng/ml), and TGF-β (Cat. No. 356039; 10 ng/ml) proteins and NA/LE Mouse Anti-Human IFN-γ (Cat. No. 554698; 10 µg/ml) antibody for 5 days. The cells were harvested and restimulated with PMA (Sigma P8139; 50 ng/ml) and ionomycin (Sigma I9657; 1 µg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724) for 5 hours. The cells were then fixed and permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeablization Kit (Cat. No. 554714) followed by staining with Alexa Fluor® 647 Mouse Anti-Human IL-9 (Cat. No. 560813) and PE Mouse Anti-Human CD4 (Cat. No. 555347) antibodies. Two-color flow cytometric dot plots showing correlated expression patterns of CD4 versus IL-9 were derived from CD4-positive cell gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry was performed on a BD LSRII™ System.
Flow cytometric analysis of IL-9 in stimulated human CD4-positive T cells. Human peripheral blood mononuclear cells were stimulated in a tissue culture plate coated with NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 µg/ml, coated overnight at 4°C) and soluble NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 1 µg/ml) antibodies plus recombinant Human IL-2 (Cat. No. 554603; 10 ng/ml), IL-4 (Cat. No. 554605; 50 ng/ml), and TGF-β (Cat. No. 356039; 10 ng/ml) proteins and NA/LE Mouse Anti-Human IFN-γ (Cat. No. 554698; 10 µg/ml) antibody for 5 days. The cells were harvested and restimulated with PMA (Sigma P8139; 50 ng/ml) and ionomycin (Sigma I9657; 1 µg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724) for 5 hours. The cells were then fixed and permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeablization Kit (Cat. No. 554714) followed by staining with Alexa Fluor® 647 Mouse Anti-Human IL-9 (Cat. No. 560813) and PE Mouse Anti-Human CD4 (Cat. No. 555347) antibodies. Two-color flow cytometric dot plots showing correlated expression patterns of CD4 versus IL-9 were derived from CD4-positive cell gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry was performed on a BD LSRII™ System.
Product Details
BD Pharmingen™
IL9; IL-9; interleukin-9; HP40; P40
Human (QC Testing)
Mouse C57BL/6 IgG1, κ
Human IL-9 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2033983
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO
Preparation And Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Fixation and Permeabilization Solution RUO
Size
125 mL
Cat No.
554722
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Fixation/Permeabilization Kit RUO
Size
250 Tests
Cat No.
554714
Antibody Details
MH9A3
The MH9A3 monoclonal antibody specifically binds to human interleukin-9 (IL-9). Human IL-9 is a multifunctional cytokine and a member of the type I cytokine (hematopoietin) family that includes IL-2, IL-4, IL-7, IL-15 and IL-21. This cytokine is encoded by the IL9 gene that is resident on chromosome 5q31.1. IL-9 is expressed by activated CD4-positive T helper cells, by some transformed T cells and by eosinophils, mast cells and neutrophils. IL-9 induces the proliferation, differentiation, and effector function of various cell types including T lymphocytes, B lymphocytes, mast cells, eosinophils, neutrophils, hematopoietic cells and epithelial cells. It potentiates the interleukin-4-induced IgM, IgG and IgE responses by human B lymphocytes. IL-9 has been implicated in human allergic disorders such as asthma and malignancies such as Hodgkin's disease. IL-9 exerts its biological activities through binding to the surface IL-9 receptor (IL-9R) complex comprised of the IL-9R alpha subunit (IL-9Rα; CD129) and the common cytokine receptor gamma subunit (γc; CD132). IL-9 signaling through its receptor includes activation of the Janus kinases 1 and 3 ( JAK1 and JAK3) and activation of Signal transducer and activator of transcription 1, 3 and 5 factors (STAT1, STAT3 and STAT5).
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
Citations & References
Development References (9)
-
Demoulin JB, Van Roost E, Stevens M, Groner B, Renauld JC. Distinct roles for STAT1, STAT3, and STAT5 in differentiation gene induction and apoptosis inhibition by interleukin-9. J Biol Chem. 1999; 274:25855-25861. (Biology). View Reference
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Dugas B, Renauld JC, Pène J, et al. Interleukin-9 potentiates the interleukin-4-induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes. Eur J Immunol. 1993 July; 23(7):1687-1692. (Biology). View Reference
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Houssiau FA, Schandene L, Stevens M. A cascade of cytokines is responsible for IL-9 expression in human T cells. Involvement of IL-2, IL-4, and IL-10. J Immunol. 1995; 154(6):2624-2630. (Biology). View Reference
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Jenmalm MC, Van Snick J, Cormont F, Salman B. Allergen-induced Th1 and Th2 cytokine secretion in relation to specific allergen sensitization and atopic symptoms in children. Clin Exp Allergy. 2001 October; 31(10):1528-1535. (Clone-specific: ELISA). View Reference
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Knoops L, Renauld JC. IL-9 and its receptor: from signal transduction to tumorigenesis. Growth Factors. 2004; 22:207-215. (Biology). View Reference
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Merz H, Houssiau FA, Orscheschek K, et al. Interleukin-9 expression in human malignant lymphomas: unique association with Hodgkin's disease and large cell anaplastic lymphoma. Blood. 1991 September; 78(5):1311-1317. (Biology). View Reference
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Renauld JC. New insights into the role of cytokines in asthma. J Clin Pathol. 2001 August; 54(8):577-589. (Biology). View Reference
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Soler D, Chapman TR, Poisson LR. CCR8 expression identifies CD4 memory T cells enriched for FOXP3+ regulatory and Th2 effector lymphocytes. J Immunol. 2006; 177(10):6940-6951. (Biology). View Reference
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Soroosh P, Doherty TA. Th9 and allergic disease. Immunology. 2009; 127(4):450-458. (Biology). View Reference
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.
