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BV650 Rat Anti-Mouse IL-10
BV650  Rat Anti-Mouse IL-10
Two-color flow cytometric analysis of IL-10 expression in stimulated mouse T cells. Splenic CD4+ T cells were cultured (2 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 25 μg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml) antibodies and with Recombinant IL-2 (Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 25 ng/ml). The stimulated cells were subsequently cultured (3 days) with Recombinant IL-2 and IL-4. The cells were then stimulated with PMA (Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). After 6 hours, the cells were harvested, washed, 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 BD Horizon™ BV650 Rat IgG2b, κ Isotype Control (Cat. No. 563233; Left Panel) or BD Horizon BV650 Rat Anti-Mouse IL-10 antibody (Cat. No. 564083; Right Panel). Flow cytometric contour plots showing correlated expression of IL-10 (or Ig Isotype control staining) versus Autofluorescence were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.
Two-color flow cytometric analysis of IL-10 expression in stimulated mouse T cells. Splenic CD4+ T cells were cultured (2 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 25 μg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml) antibodies and with Recombinant IL-2 (Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 25 ng/ml). The stimulated cells were subsequently cultured (3 days) with Recombinant IL-2 and IL-4. The cells were then stimulated with PMA (Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). After 6 hours, the cells were harvested, washed, 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 BD Horizon™ BV650 Rat IgG2b, κ Isotype Control (Cat. No. 563233; Left Panel) or BD Horizon BV650 Rat Anti-Mouse IL-10 antibody (Cat. No. 564083; Right Panel). Flow cytometric contour plots showing correlated expression of IL-10 (or Ig Isotype control staining) versus Autofluorescence were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.
Product Details
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BD Horizon™
Interleukin-10; Il10; CSIF; Cytokine synthesis inhibitory factor
Mouse (QC Testing)
Rat IgG2b
Recombinant mouse IL-10
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_2738583
Aqueous buffered solution containing BSA and ≤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 BD Horizon™ BV650 under optimum conditions, and unconjugated antibody and free BD Horizon™ BV650 were removed.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. 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.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  6. Brilliant Violet™ 650 is a trademark of Sirigen.
  7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
564083 Rev. 1
Antibody Details
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JES5-16E3

The JES5-16E3 monoclonal antibody specifically binds to the mouse cytokine, Interleukin-10 (IL-10). IL-10 is also known as Cytokine Synthesis Inhibitory Factor (CSIF). It is produced by various activated cell types including CD4+ T cells, CD8+ T cells, T regulatory cells, NK T cells, B1 B cells, NK cells, macrophages, dendritic cells, mast cells, granulocytes and keratinocytes. IL-10 plays a pivotal role in regulating immune responses and protecting the host from damage caused by inflammatory and autoimmune responses. IL-10 has numerous biological activities including the inhibition of cytokine synthesis by activated T cells, NK cells, monocytes, and macrophages. In the presence of accessory cells, IL-10 inhibits mitogen- or anti-CD3 induced proliferation of T lymphocytes.  IL-10 has also been shown to costimulate the development of thymocytes, B cell differentiation and the generation of cytotoxic T cells. The immunogen used to generate the JES5-16E3 hybridoma was recombinant mouse IL-10. JES5-16E3 is a neutralizing antibody.

The antibody was conjugated to BD Horizon™ BV650 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 650-nm. BD Horizon BV650 can be excited by the violet laser and detected in a filter used to detect APC-like dyes (eg, 660/20-nm filter).  Due to the excitation and emission characteristics of the acceptor dye, there will be  spillover into the APC and Alexa Fluor® 700 detectors. However, the spillover can be corrected through compensation as with any other dye combination.

564083 Rev. 1
Format Details
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BV650
The BD Horizon Brilliant Violet™ 650 (BV650) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This tandem fluorochrome is comprised of a BV421 donor with an excitation maximum (Ex Max) of 406-nm and an acceptor dye with an emission maximum (Em Max) at 649-nm. BV650, driven by BD innovation, is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 650-nm (e.g., a 660/20-nm bandpass filter). The acceptor dye can be excited by the Red (628–640-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.
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BV650
Violet 405 nm
406 nm
649 nm
564083 Rev.1
Citations & References
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Development References (4)

  1. Andersson U, Andersson J. Immunolabeling of cytokine-producing cells in tissues and in suspension. In: Fradelizie D, Emelie D, ed. Cytokine Producing Cells. Paris: Inserm; 1994:32-49.
  2. Litton MJ, Sander B, Murphy E, O'Garra A, Abrams JS. Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J Immunol Methods. 1994; 175(1):47-58. (Clone-specific: Neutralization). View Reference
  3. 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
  4. 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. (Clone-specific: Immunocytochemistry (cytospins), Neutralization). View Reference
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564083 Rev. 1

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.