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Two color flow cytometric analysis of IL-2 expression by stimulated mouse splenocytes. Mouse splenic leucocytes were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139; 50 ng/ml) and Ionomycin (Sigma I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested and washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with FITC Rat Anti-Mouse CD4 antibody (Cat. No. 553046/553047/561835) and either BD Horizon™ BV605 Rat IgG2b, κ Isotype Control (Cat. No. 563145; Left Panel) or BD Horizon™ BV605 Rat Anti-Mouse IL-2 antibody (Cat. No. 563911; Right Panel) by using BD Biosciences Intracellular Cytokine Staining protocol. Flow cytometric dot plots showing correlated expression of IL-2 (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated leucocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.


BD Horizon™ BV605 Rat Anti-Mouse IL-2

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Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
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.
- 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.
- Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from BD Horizon™ BV421 may be observed. Therefore, we recommend that individual compensation controls be performed for every BD Horizon™ BV605 conjugate.
- CF™ is a trademark of Biotium, Inc.
- 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.
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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.
This antibody is conjugated to BD Horizon BV605 which is part of the BD Horizon Brilliant™ Violet family of dyes. With an Ex Max of 407-nm and Em Max of 602-nm, BD Horizon BV605 can be excited by a violet laser and detected with a standard 610/20-nm filter set. BD Horizon BV605 is a tandem fluorochrome of BD Horizon BV421 and an acceptor dye with an Em max at 605-nm. Due to the excitation of the acceptor dye by the green (532 nm) and yellow-green (561 nm) lasers, there will be significant spillover into the PE and BD Horizon PE-CF594 detectors off the green or yellow-green lasers. BD Horizon BV605 conjugates are very bright, often exhibiting brightness equivalent to PE conjugates and can be used as a third color off of the violet laser.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794).

Development References (4)
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Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
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Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific: ELISA, Immunoprecipitation). 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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Sarder M, Saito H, Abe K. Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain. Brain Res. 1993; 625(2):347-350. (Clone-specific: ELISA). View Reference
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