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APC-R700 Rat Anti-Human IL-10
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APC-R700 Rat Anti-Human IL-10

Multiparameter flow cytometric analysis of IL-10 expressed in stimulated human lymphocytes. An enriched population of CD4+ peripheral blood mononuclear cells (PBMC) was obtained by panning using plate-bound Purified NA/LE Mouse Anti-Human CD4 antibody (Cat. No. 555343). The CD4+ PBMC were cultured (5 d) with plate-bound NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 μg/ml, coated overnight at 4°C) and soluble NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 1 μg/ml) antibodies plus recombinant Human IL-2 (Cat. No. 554603; 20 ng/ml) and IL-4 (Cat. No. 554605; 40 ng/ml) proteins. The cells were restimulated (5 h) with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P8139; 50 ng/ml) and ionomycin (Sigma I9657; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724). The cells were harvested, fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were stained in BD Perm/Wash™ Buffer with BD Horizon™ BV421 Mouse Anti- Human CD4 antibody (Cat. No. 565997/566907) and either BD Horizon™ APC-R700 Rat IgG2a, κ Isotype Control (Cat. No. 564982; Left Plot) or BD Horizon APC-R700 Mouse Anti-Human IL-10 antibody (Cat. No. 567012/567013; Right Plot). A bivariate pseudocolor density plot showing the coexpressed levels of IL-10 (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.

Multiparameter flow cytometric analysis of IL-10 expressed in stimulated human lymphocytes. An enriched population of CD4+ peripheral blood mononuclear cells (PBMC) was obtained by panning using plate-bound Purified NA/LE Mouse Anti-Human CD4 antibody (Cat. No. 555343). The CD4+ PBMC were cultured (5 d) with plate-bound NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 μg/ml, coated overnight at 4°C) and soluble NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 1 μg/ml) antibodies plus recombinant Human IL-2 (Cat. No. 554603; 20 ng/ml) and IL-4 (Cat. No. 554605; 40 ng/ml) proteins. The cells were restimulated (5 h) with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P8139; 50 ng/ml) and ionomycin (Sigma I9657; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724). The cells were harvested, fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were stained in BD Perm/Wash™ Buffer with BD Horizon™ BV421 Mouse Anti- Human CD4 antibody (Cat. No. 565997/566907) and either BD Horizon™ APC-R700 Rat IgG2a, κ Isotype Control (Cat. No. 564982; Left Plot) or BD Horizon APC-R700 Mouse Anti-Human IL-10 antibody (Cat. No. 567012/567013; Right Plot). A bivariate pseudocolor density plot showing the coexpressed levels of IL-10 (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.

Product Details
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BD Horizon™
Interleukin-10; CSIF; Cytokine synthesis inhibitory factor; TGIF
Human (QC Testing), Viral (Tested in Development)
Rat IgG2a, κ
Recombinant Human IL-10
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2870004
Aqueous buffered solution containing BSA, protein stabilizer, glycerol 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 to the dye under optimum conditions and unconjugated antibody and free dye were removed.

Recommended Assay Procedures

BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation).  When fluorochrome conjugated antibodies are bound to CompBeads, they have spectral properties very similar to cells.   However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls.  It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. 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.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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Antibody Details
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JES3-19F1

The JES3-19F1 monoclonal antibody specifically recognizes human Interleukin-10 (IL-10) that is encoded by IL10.  IL-10 is also known as Cytokine Synthesis Inhibitory Factor (CSIF), B cell-derived T cell growth factor (B-TCGF), and T-cell growth inhibitory factor (TGIF). The JES3-19F1 antibody crossreacts with ebvIL-10 protein, the Epstein-Barr viral IL-10 homolog (viral IL-10 or vIL-10) encoded by the BCRF1 gene. IL-10 is produced by a variety of cells such as some activated T cells and B cells including regulatory T cells (Treg) and B cells (Breg), monocytes and macrophages, dendritic cells (DC), keratinocytes, and mast cells. IL-10 is a multifunctional cytokine that can downregulate immune and proinflammatory responses. IL-10 can act to reduce expression of major histocompatibility complex class II antigens, costimulatory molecules, or proinflammatory cytokines including IL-1β, IL-2, IL-3,  IL-12, IFN-γ, TNF or GM-CSF expressed by activated monocytes, macrophages, dendritic cells (DC), natural killer (NK) cells, or T cells. IL-10 has been shown to play a role in chronic viral infections. IL-10 can also enhance B cell survival, proliferation, and differentiation to become antibody-producing cells. The JES3-19F1 antibody reportedly neutralizes the biological activity of human IL-10 and ebvIL-10. IL-10 mediates its biological activities by signaling through a heterotetrameric receptor complex composed of the type II cytokine receptor subunits CD210a (IL-10 Rα) and CD210b (IL-10 Rβ).

        This antibody was conjugated to BD Horizon APC-R700, which has been developed exclusively by BD Biosciences as a better alternative to Alexa Fluor® 700. APC-R700 excites and emits at similar wavelengths to Alexa Fluor® 700 yet exhibits significantly improved brightness. This dye can be excited by the red laser and detected with the same filter set as Alexa Fluor® (eg, 730/45-nm filter).

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Format Details
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APC-R700
The BD Horizon™ APC-R700 (APC-R700) Dye is a part of the BD APC red family of dyes. This tandem fluorochrome is comprised of an Allophycocyanin (APC) dye donor that has excitation maximum (Ex Max) of 651-nm and an acceptor dye, R700, with an emission maximum (Em Max) at 706-nm. APC-R700, driven by BD innovation, is designed to be excited by the red (627–640-nm) laser and detected using an optical filter centered near 710-nm (e.g., a 720/40-nm bandpass filter). APC-R700 is a brighter alternative to Alexa Fluor™ 700. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
APC-R700
Red 627-640 nm
651 nm
706 nm
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Citations & References
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Development References (10)

  1. Andersson J, Abrams J, Bjork L, et al. Concomitant in vivo production of 19 different cytokines in human tonsils. Immunology. 1994; 83(1):16-24. (Clone-specific: Immunohistochemistry). View Reference
  2. Brodeur ND, Spencer JV. Antibodies to human IL-10 neutralize ebvIL-10-mediated cytokine suppression but have no effect on cmvIL-10 activity.. Virus Res. 2010; 153(2):265-8. (Clone-specific: Bioassay, Blocking, ELISA, Functional assay, Inhibition, Neutralization, Western blot). View Reference
  3. Cousins DJ, Lee TH, Staynov DZ. Cytokine coexpression during human Th1/Th2 cell differentiation: direct evidence for coordinated expression of Th2 cytokines.. J Immunol. 2002; 169(5):2498-506. (Clone-specific: Flow cytometry). View Reference
  4. D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993; 178(3):1041-1048. (Biology). View Reference
  5. Mielle J, Audo R, Hahne M, et al. IL-10 Producing B Cells Ability to Induce Regulatory T Cells Is Maintained in Rheumatoid Arthritis. Front Immunol. 2018; 9:961. (Clone-specific: Flow cytometry). View Reference
  6. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor.. Annu Rev Immunol. 2001; 19:683-765. (Biology). View Reference
  7. Schlaak JF, Schmitt E, Hüls C, Meyer zum Büschenfelde KH, Fleischer B. A sensitive and specific bioassay for the detection of human interleukin-10. J Immunol Methods. 1994; 168(1):49-54. (Clone-specific: Bioassay, Functional assay, Inhibition). View Reference
  8. Wehrens EJ, Mijnheer G, Duurland CL, et al. Functional human regulatory T cells fail to control autoimmune inflammation due to PKB/c-akt hyperactivation in effector cells.. Blood. 2011; 118(13):3538-48. (Clone-specific: Flow cytometry). View Reference
  9. Yin Y, Mitson-Salazar A, Prussin C. Detection of Intracellular Cytokines by Flow Cytometry. Curr Protoc Immunol. 2015; 110:6.24.21-26.24.18. (Methodology: Flow cytometry). View Reference
  10. Zielinski CE, Mele F, Aschenbrenner D, et al. Pathogen-induced human TH17 cells produce IFN-gamma or IL-10 and are regulated by IL-1beta. Nature. 2012; 484(7395):514-518. (Clone-specific: Flow cytometry). View Reference
View All (10) View Less
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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.