Two-color flow cytometric analysis of IL-2 expressed by activated CD4-positive splenocytes. Mouse BALB/c spleen cells were activated for 4 hours using Leukocyte Activation Cocktail, with BD GolgiPlug™ (Cat. No. 550583) that contains Phorbol 12-Myristate 13-Acetate (PMA), ionomycin and brefeldin A. The cells were then fixed and permeabilized using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Perm/Wash™ Buffer (Cat. No. 554723). The permeabilized cells were stained with APC Rat Anti-Mouse CD4 (Cat. No. 553051/561091) and either Alexa Fluor® 700 Rat Anti-Mouse IL-2 (Cat No. 561287, Left Panel) or Alexa Fluor® 700 Rat IgG2b Isotype Control (Cat No.557964, 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 IL-2-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IL-2 (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.
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Two-color flow cytometric analysis of IL-2 expressed by activated CD4-positive splenocytes. Mouse BALB/c spleen cells were activated for 4 hours using Leukocyte Activation Cocktail, with BD GolgiPlug™ (Cat. No. 550583) that contains Phorbol 12-Myristate 13-Acetate (PMA), ionomycin and brefeldin A. The cells were then fixed and permeabilized using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Perm/Wash™ Buffer (Cat. No. 554723). The permeabilized cells were stained with APC Rat Anti-Mouse CD4 (Cat. No. 553051/561091) and either Alexa Fluor® 700 Rat Anti-Mouse IL-2 (Cat No. 561287, Left Panel) or Alexa Fluor® 700 Rat IgG2b Isotype Control (Cat No.557964, 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 IL-2-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IL-2 (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™
Il2; Interleukin-2; T-cell growth factor; TCGF
Mouse (QC Testing)
Rat IgG2b
Mouse IL-2 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
16183
AB_10679118
Aqueous buffered solution containing protein stabilizer and ≤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® 700 under optimum conditions, and unreacted Alexa Fluor® 700 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures
Flow cytometry: The JES6-5H4 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-2 producing cells within mixed cell populations. A useful control investigators may consider using for demonstrating specificity of staining, is to pre-block with one of the following reagents: (1) recombinant mouse IL-2 (Cat. No. 550069) or (2) unlabeled JES6-5H4 antibody (Cat. No. 554425), prior to staining.
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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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® 700 has an adsorption maximum of ~700nm and a peak fluorescence emission of ~720nm. Before staining cells with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Alexa Fluor® 700 Rat IgG2b, κ Isotype Control RUO
Size
0.1 mg
Cat No.
557964
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Leukocyte Activation Cocktail, with BD GolgiPlug™ RUO
Size
200 µL
Cat No.
550583
APC Rat Anti-Mouse CD4 RUO
Size
0.1 mg
Cat No.
553051
561287 Rev. 1
Antibody Details
JES6-5H4
The JES6-5H4 monoclonal antibody specifically binds to mouse interleukin-2 (IL-2), a multifunctional cytokine that plays pivotal roles in immunity and tolerance. It is produced by activated T cells and affects the activation, growth, proliferation and/or differentiation of various cell types including T and B lymphocytes and their precursors, LAK cells, NK cells, and monocytes/macrophages. IL-2 mediates its biological activities by binding to IL-2 receptor complexes. The intermediate affinity IL-2R is comprised of IL-2Rβ (CD122) and common gamma chain (γc; CD132) subunits, whereas the high-affinity IL-2R is comprised of IL-2Rα (CD25), IL-2Rβ, and γc subunits. The JES6-5H4 monoclonal antibody binds to IL-2 and neutralizes its biological activity.
561287 Rev. 1
Format Details
Alexa Fluor™ 700
Alexa Fluor™ 700 dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 697 nm and an emission maximum (Em Max) at 719-nm. Alexa Fluor™ 700 is designed to be excited by the Red (627–640-nm) laser and detected using an optical filter centered near 720-nm (e.g., a 720/40-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 700
Red 627-640 nm
697 nm
719 nm
561287 Rev.1
Citations & References
Development References (12)
-
Awatsuji H, Furukawa Y, Nakajima M, Furukawa S, Hayashi K.. Interleukin-2 as a neurotrophic factor for supporting the survival of neurons cultured from various regions of fetal rat brain. J Neurosci Res. 1993; 35(3):305-311. (Biology). View Reference
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Gillis S, Ferm MM, Ou W, Smith KA. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978; 120(6):2027-2032. (Biology). View Reference
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Gillis S, Ferm MM, Ou W, Smith KA. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978; 120(6):2027-2032. (Biology). View Reference
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Helms T, Boehm BO, Asaad RJ, Trezza RP, Lehmann PV, Tary-Lehmann M. Direct visualization of cytokine-producing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J Immunol. 2000; 164(7):3723-3732. (Biology). View Reference
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Kashima N, Nishi-Takaoka C, Fujita T, et al. Unique structure of murine interleukin-2 as deduced from cloned cDNAs. Nature. 1985; 313(6001):402-404. (Biology). View Reference
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Kubo M, Cinader B. Polymorphism of age-related changes in interleukin (IL) production: differential changes of T helper subpopulations, synthesizing IL 2, IL 3 and IL 4. Eur J Immunol. 1990; 20(6):1289-1296. (Biology). View Reference
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Mochizuki DY, Watson J, Gillis S. Biochemical separation of interleukin 2. J Immunol Methods. 1980; 39(3):185-201. (Biology). View Reference
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Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986; 136(7):2348-2357. (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: ELISA). View Reference
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Watson J, Mochizuki D. Interleukin 2: a class of T cell growth factors. Immunol Rev. 1980; 51:257-278. (Biology). View Reference
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Yokota T, Arai N, Lee F, Rennick D, Mosmann T, Arai K. Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells. Proc Natl Acad Sci U S A. 1985; 82(1):68-72. (Biology). View Reference
561287 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.
