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      PE Rat Anti-Human and Viral IL-10
      PE Rat Anti-Human and Viral IL-10
      Expression of IL-10 by stimulated human monocytes. Human PBMC were stimulated overnight with 1.0 µg/ml LPS in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were then harvested fixed, permeabilized, and subsequently stained with 20 µl of PE-rat anti-human IL-10 antibody (Cat. No. 554498) following the Usage section above and BD Pharmingen staining protocol (middle panel). The data reflect gating on monocytes, based on forward and side light scatter. To demonstrate specificity of staining, the binding of PE-JES3-9D7 was blocked by the preincubation of the fixed/permeabilized cells with the unlabeled JES3-9D7 antibody (5.0 µg final concentration; Cat. No. 554496; right panel) prior to staining with the PE-JES3-9D7 antibody. The quadrant markers for the bivariate dot plots were set based on the staining profile using 20µl of PE-R3-34 isotype control antibody (Cat. No. 559318, left panel) and verified using the unlabeled antibody blocking specificity control.
      PE Rat Anti-Human and Viral IL-10
      Expression of IL-10 by stimulated human monocytes. Human PBMC were stimulated overnight with 1.0 µg/ml LPS in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were then harvested fixed, permeabilized, and subsequently stained with 20 µl of PE-rat anti-human IL-10 antibody (Cat. No. 554498) following the Usage section above and BD Pharmingen staining protocol (middle panel). The data reflect gating on monocytes, based on forward and side light scatter. To demonstrate specificity of staining, the binding of PE-JES3-9D7 was blocked by the preincubation of the fixed/permeabilized cells with the unlabeled JES3-9D7 antibody (5.0 µg final concentration; Cat. No. 554496; right panel) prior to staining with the PE-JES3-9D7 antibody. The quadrant markers for the bivariate dot plots were set based on the staining profile using 20µl of PE-R3-34 isotype control antibody (Cat. No. 559318, left panel) and verified using the unlabeled antibody blocking specificity control.
      Product Details
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      BD Pharmingen™
      Human (QC Testing), Viral (Tested in Development)
      Rat IgG1
      Recombinant Human IL-10
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      AB_397232
      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. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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      Recommended Assay Procedures

      Immunofluorescent Staining and Flow Cytometric Analysis: The PE-conjugated JES3-9D7 antibody can be used for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-10-producing cells within mixed cell populations (see image). This 100 Test Size formulation of the PE-conjugated JES3-9D7 antibody has been pre-titrated to assure effective intracellular detection of human and viral IL-10 using 20 µl/1 x 10^6 cells. A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated JES3-9D7 antibody with ligand (recombinant human IL-10; Cat. No. 554611) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled JES3-9D7 antibody (Cat. No. 554496) prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is also available in a 100 Test Size formulation PE-R3-34 (Cat. No. 559318). For specific methodology, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.

      Important Note: This pretitered antibody solution does not contain a cell permeabilization agent. It is necessary to include a cell permeabilization agent when using the pre-titered antibody solution to stain fixed and permeabilized cells. Perm/Wash™ Buffer (Cat. No.554723) contains the permeabilization agent saponin and is useful for this purpose as described in the USAGE section below.

      USAGE

      1. Resuspend 1 x 10^6 fixed and permeabilized cells in 20 µl of the pre-titered antibody solution and 30 µl of 1X Perm/Wash™ Buffer (Cat. No. 554723).

      2. Incubate the cell suspension for 15 minutes (at RT or 4°C).

      3. Wash twice in 100 µl of 1X Perm/Wash™ Buffer (Cat. No. 554723).

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      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. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      6. 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.
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      559337 Rev. 3
      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.

      559337 Rev. 3
      Format Details
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      PE
      R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      altImg
      PE
      Yellow-Green 488 nm, 532 nm, 561 nm
      496 nm, 566 nm
      576 nm
      559337 Rev.3
      Citations & References
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      View product citations for antibody "559337" on CiteAb

      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: Blocking, Flow cytometry). 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
      View All (6) View Less
      559337 Rev. 3

       

      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.