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      PE Rat Anti-Mouse IL-6
      PE Rat Anti-Mouse IL-6
      Expression of IL-6 by stimulated BALB/c cells. BALB/c bone marrow cells were cultured for 2 weeks in the presence of recombinant mouse GM-CSF (20 ng/ml final concentration; Cat. No. 554586). The cells were washed and stimulated with recombinant mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587) for 4 hours followed by an overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. #L-8272) and GolgiPlug™ (1 µg/ml final concentration; Cat. No. 555029; aka Brefeldin A). Adherent cells were incubated with 1x trypsin EDTA at 37°C for 10 minutes and gently dislodged by pipetting for cell harvest. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block™ (10 µg/ml final concentration; Cat. No. 553142). The cells were fixed , permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody (PE-MP5-20F3, Cat. No. 554401) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-MP5-20F3 antibody was blocked by preincubation of the PE-MP5-20F3 antibody with recombinant mouse IL-6 (0.12 µg; Cat. No. 554582; right panel) and by preincubation of the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (5 µg; Cat. No. 554400; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
      PE Rat Anti-Mouse IL-6
      Expression of IL-6 by stimulated BALB/c cells. BALB/c bone marrow cells were cultured for 2 weeks in the presence of recombinant mouse GM-CSF (20 ng/ml final concentration; Cat. No. 554586). The cells were washed and stimulated with recombinant mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587) for 4 hours followed by an overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. #L-8272) and GolgiPlug™ (1 µg/ml final concentration; Cat. No. 555029; aka Brefeldin A). Adherent cells were incubated with 1x trypsin EDTA at 37°C for 10 minutes and gently dislodged by pipetting for cell harvest. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block™ (10 µg/ml final concentration; Cat. No. 553142). The cells were fixed , permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody (PE-MP5-20F3, Cat. No. 554401) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-MP5-20F3 antibody was blocked by preincubation of the PE-MP5-20F3 antibody with recombinant mouse IL-6 (0.12 µg; Cat. No. 554582; right panel) and by preincubation of the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (5 µg; Cat. No. 554400; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
      Product Details
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      BD Pharmingen™
      Mouse (QC Testing)
      Rat IgG1
      Mouse IL-6 Recombinant Protein
      Intracellular staining (flow cytometry) (Routinely Tested)
      0.2 mg/ml
      15978,16193
      AB_395367
      Aqueous buffered solution containing ≤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 MP5-20F3 antibody (Cat. No. 554401) can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate IL-6-producing cells within mixed cell populations (see images). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.25 µg mAb/million cells)  For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook.  A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated MP5-20F3 antibody with ligand (e.g., recombinant mouse IL-6; Cat No. 554582) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (Cat. No. 554400) 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 mouse or human cells is purified R3-34 (Cat. No. 554685); use at comparable concentrations to antibody of interest (e.g., ≤ 0.25 µg mAb/1 million cells).

      Other applications for this clone in additional formats available are:

      Neutralization: The NA/LE™ MP5-20F3 antibody (Cat. No. 554398) is useful for neutralization of mouse IL-6 bioactivity.

      ELISA Capture: The purified MP5-20F3 antibody (Cat. No. 554401) is useful as a capture antibody for a sandwich ELISA for measuring mouse IL-6 protein levels with the biotinylated detection antibody (Cat. No. 554402) and recombinant mouse IL-6 (Cat. No. 554582) as a standard.

      Western Blot: The MP5-20F3 antibody has been reported to be useful for Western blotting. Please note that this application is not tested at BD Biosciences Pharmingen.

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      Product Notices

      1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      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.
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      554401 Rev. 1
      Antibody Details
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      MP5-20F3

      The MP5-20F3 monoclonal antibody specifically binds to mouse interleukin-6 (IL-6). The immunogen used to generate the MP5-20F3 hybridoma was recombinant mouse IL-6.

      554401 Rev. 1
      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
      554401 Rev.1
      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, 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, IC/FCM Block). 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: ELISA, Flow cytometry). View Reference
      5. Starnes HF Jr, Pearce MK, Tewari A, Yim JH, Zou JC, Abrams JS. Anti-IL-6 monoclonal antibodies protect against lethal Escherichia coli infection and lethal tumor necrosis factor-alpha challenge in mice. J Immunol. 1990; 145(12):4185-4191. (Clone-specific: Neutralization). View Reference
      6. Suda T, O'Garra A, MacNeil I, Fischer M, Bond MW, Zlotnik A. Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas. Cell Immunol. 1990; 129(1):228-240. (Clone-specific: Neutralization). View Reference
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      554401 Rev. 1

      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.

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