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BV786 Rat Anti-Human and Viral IL-10
BV786 Rat Anti-Human and Viral IL-10

Two-color flow cytometric analysis of IL-10 expression in stimulated human lymphocytes. Human peripheral blood mononuclear cells were stimulated with immobilized Purified NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 µg/ml for plate coating) and soluble Purified NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 2 µg/ml) antibodies and Recombinant Human IL-2 (Cat. No. 554603; 10 ng/ml) and Human IL-4 (Cat. No. 554605; 25 ng/ml) for 2 days. The stimulated cells were washed and cultured in medium containing IL-2 and IL-4 for another 3 days. Finally, the cells were harvested and restimulated for 5 hr with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724).

       The cells were harvested, 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 either BD Horizon™ BV786 Rat IgG1 κ Isotype Control (Cat. No. 563847; Left Panel) or BD Horizon™ BV786 Mouse Anti-Human IL-10 and Viral IL-10 antibody (Cat. No. 564049; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot plots show the correlated expression patterns of IL-10 (or Ig Isotype control staining) versus autofluorescence for gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Two-color flow cytometric analysis of IL-10 expression in stimulated human lymphocytes. Human peripheral blood mononuclear cells were stimulated with immobilized Purified NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 µg/ml for plate coating) and soluble Purified NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 2 µg/ml) antibodies and Recombinant Human IL-2 (Cat. No. 554603; 10 ng/ml) and Human IL-4 (Cat. No. 554605; 25 ng/ml) for 2 days. The stimulated cells were washed and cultured in medium containing IL-2 and IL-4 for another 3 days. Finally, the cells were harvested and restimulated for 5 hr with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724).

       The cells were harvested, 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 either BD Horizon™ BV786 Rat IgG1 κ Isotype Control (Cat. No. 563847; Left Panel) or BD Horizon™ BV786 Mouse Anti-Human IL-10 and Viral IL-10 antibody (Cat. No. 564049; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot plots show the correlated expression patterns of IL-10 (or Ig Isotype control staining) versus autofluorescence for gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Product Details
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BD Horizon™
CSIF; Cytokine synthesis inhibitory factor; TGIF
Human (QC Testing), Viral (Tested in Development)
Rat IgG1
Recombinant Human IL-10
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2738563
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™ BV786 under optimum conditions, and unconjugated antibody and free BD Horizon™ BV786 were removed.

Recommended Assay Procedures

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. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).

Product Notices

  1. 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).
  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. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  8. Cy is a trademark of GE Healthcare.
  9. BD Horizon Brilliant Violet 786 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,354,239.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
564049 Rev. 2
Antibody Details
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JES3-9D7

The JES3-9D7 monoclonal antibody specifically reacts with human IL-10 (Interleukin-10) and viral IL-10. The immunogen used to generate the JES3-9D7 hybridoma was recombinant human IL-10 expressed by COS cells. IL-10 is also known as CSIF (Cytokine synthesis inhibitory factor) and (TGIF) T-cell growth inhibitory factor. IL-10 is expressed by various cell types including activated monocytes, macrophages, dendritic cells, mast cells, granulocytes, and lymphocytes. IL-10 is a pleiotropic cytokine that can downregulate proinflammatory immune responses, such as Th1-like responses, while promoting other responses including B cell proliferation and antibody production.

The antibody was conjugated to BD Horizon BV786 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 786-nm.  BD Horizon BV786 can be excited by the violet laser and detected in a filter used to detect Cy™7-like dyes (eg, 780/60-nm filter).  

564049 Rev. 2
Format Details
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BV786
The BD Horizon Brilliant Violet™ 786 (BV786) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This tandem fluorochrome is comprised of a BV421 donor with an Ex Max of 407-nm and an acceptor dye with an Em Max at 786-nm.  BV786, driven by BD innovation, is designed to be excited by the violet laser and detected using a filter, centered near 785 nm (e.g. 780/60 nm bandpass filter).  Please ensure that your instrument’s configurations (lasers and filters) are appropriate for this dye.
altImg
BV786
Violet 405 nm
407 nm
786 nm
564049 Rev.2
Citations & References
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Development References (6)

  1. 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
  2. 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). View Reference
  3. Burdin N, Peronne C, Banchereau J, Rousset F. Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10. J Exp Med. 1993; 177(2):295-304. (Clone-specific: ELISA). View Reference
  4. Gotlieb WH, Abrams JS, Watson JM, Velu TJ, Berek JS, Martinez-Maza O. Presence of interleukin 10 (IL-10) in the ascites of patients with ovarian and other intra-abdominal cancers. Cytokine. 1992; 4(5):385-390. (Clone-specific: ELISA). View Reference
  5. 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: IC/FCM Block). View Reference
  6. Yssel H, De Waal Malefyt R, Roncarolo MG, et al. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J Immunol. 1992; 149(7):2378-2384. (Clone-specific: ELISA). View Reference
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564049 Rev. 2

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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.