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Two-color flow cytometric analysis of IL-6 expression in stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were primed for 2 hr with Recombinant Human IFN-γ (10 ng/ml; Cat. No. 554617/554616) and stimulated with lipopolysaccharide (1.0 µg/ml; Sigma, Cat. No. L-8274; 6 hr) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing monensin) (Cat. No. 554724). The PBMC were then stained with APC Mouse Anti-Human CD14 antibody (Cat. No. 555399/561708/561383). After washing with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), the cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722). The cells were then stained with either BD Horizon™ BV421 Rat IgG1, κ Isotype Control (Cat. No. 562868; Left Panel) or BD Horizon™ BV421 Anti-Human IL-6 antibody (Cat. No. 563279; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot plots show the correlated expression patterns of IL-6 (or Ig Isotype control staining) versus CD14 for gated events with the forward and side light-scatter characteristics of intact stimulated PBMC. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
BD Horizon™ BV421 Rat Anti-Human IL-6
Regulatory Status Legend
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
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- An isotype control should be used at the same concentration as the antibody of interest.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- Brilliant Violet™ 421 is a trademark of Sirigen.
Companion Products
The MQ2-13A5 monoclonal antibody specifically binds to human interleukin-6 (IL-6). IL-6 is a multifunctional cytokine that plays a central role in host defense mechanisms, including hematopoiesis, immune responses (eg, T and B cell activation and differentiation) and acute phase reactions. IL-6 can be expressed by a variety of cells including monocytes/macrophages, eosinophils, fibroblasts, vascular endothelial cells, bone marrow stromal cells, mesangial cells, hepatocytes, keratinocytes, astrocytes, T lymphocytes, B lymphocytes, and various tumor cells. IL-6 production is upregulated by cells in response to bacterial products such as lipopolysaccharide, viruses and other pro-inflammatory cytokines such as IL-1, TNF , and IFN-γ. IL-6 transcription is downregulated by cells in response to IL-4 and IL-10. The functional IL-6 Receptor (IL-6R) complex consists of two transmembrane glycoproteins, an 80-kDa low-affinity ligand-binding receptor subunit (IL-6Rα/CD126) and a 130 kDa (gp130/CD130) subunit that binds to IL-6-IL-6Rα to form the high-affinity signal transducing complex. Abnormal expression of IL-6 is related to the pathogenesis of many diseases including neoplastic (eg, multiple myeloma) and autoimmune diseases (eg, rheumatoid arthritis). The immunogen used to generate this hybridoma was COS-7 -expressed recombinant human IL-6.
The antibody was conjugated to BD Horizon™ BV421 which is part of the BD Horizon™ Brilliant Violet™ family of dyes. With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon™ BV421 can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon™ BV421 conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue™ conjugates.
Development References (7)
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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, Neutralization). 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, Neutralization). View Reference
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Gaines Das RE, Poole S. The international standard for interleukin-6. Evaluation in an international collaborative study. J Immunol Methods. 1993; 160(2):147-153. (Clone-specific: ELISA, Neutralization). View Reference
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Hirano T, Yasukawa K, Harada H, et al.. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature. 1986; 324(6092):73-76. (Biology). View Reference
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Matsuda T, Hirano T. IL-6. In: Oppenheim JJ, Feldmann M, Durum SK, Hirano T, Vilcek J, Nicola NA, ed. Cytokine Reference : A compendium of cytokines and other mediators of host defense. San Diego: Academic Press; 2001:537-563.
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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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Rincon M. Interleukin-6: from an inflammatory marker to a target for inflammatory diseases. Trends Immunol. 2012; 33(11):571-577. (Biology). View Reference
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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.