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      PE Mouse Anti-Human TNF
      PE Mouse Anti-Human TNF
      Expression of TNF protein by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma, Cat. #P-8139), calcium ionophore A23187 (0.5 µg/ml final concentration; Sigma, Cat. #C-9275) and PHA (Gibco BRL, Cat. #10576-015) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554715). The PBMC were stained with 0.25 µg PE-Cy™5 Mouse Anti-Human CD3 (Cat. No. 555334) and subsequently stained with 20 µl of PE Mouse Anti-Human TNF (Cat. No. 559321/554513/562083) by following the recommended assay procedure (left panel). To demonstrate specificity of staining, the binding of PE-MAb11 was blocked by preincubation of the conjugate with excess (1 µg) recombinant human TNF (Cat. No. 554618; middle panel) and by preincubation of the fixed/permeabilized cells with an excess of Purified Mouse Anti-Human TNF (Cat. No. 554510; right panel) prior to staining with the PE-MAb11 antibody. The quadrant markers for the bivariate dot plot was set based on the negative staining controls using isotype-matched Ig controls.
      PE Mouse Anti-Human TNF
      Expression of TNF protein by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma, Cat. #P-8139), calcium ionophore A23187 (0.5 µg/ml final concentration; Sigma, Cat. #C-9275) and PHA (Gibco BRL, Cat. #10576-015) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554715). The PBMC were stained with 0.25 µg PE-Cy™5 Mouse Anti-Human CD3 (Cat. No. 555334) and subsequently stained with 20 µl of PE Mouse Anti-Human TNF (Cat. No. 559321/554513/562083) by following the recommended assay procedure (left panel). To demonstrate specificity of staining, the binding of PE-MAb11 was blocked by preincubation of the conjugate with excess (1 µg) recombinant human TNF (Cat. No. 554618; middle panel) and by preincubation of the fixed/permeabilized cells with an excess of Purified Mouse Anti-Human TNF (Cat. No. 554510; right panel) prior to staining with the PE-MAb11 antibody. The quadrant markers for the bivariate dot plot was set based on the negative staining controls using isotype-matched Ig controls.
      Product Details
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      BD Pharmingen™
      Tumor necrosis factor alpha; TNF-a; TNF-α; TNFSF2; Cachectin
      Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
      Mouse IgG1, κ
      Recombinant Human TNF
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      AB_397219
      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 R-PE under optimum conditions, and unconjugated antibody and free PE were removed.
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      Recommended Assay Procedures

      Immunofluorescent Staining and Flow Cytometric Analysis: The PE conjugated MAb11 antibody is useful for intracellular immunofluorescent staining and flow cytometric analysis to identify and enumerate TNF producing cells within mixed cell populations (see Figure). This 100 Test Size formulation of the PE-conjugated MAb11 antibody has been pre-titrated to assure effective intracellular detection of human TNF using 20 µl/1 x 10^6 cells. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit the protocols section under "Cytokines (Intracellular Staining)" and "Intracellular Flow" posted on our web site, http://www.bdbiosciences.com/us/s/resources.

      Important Note: This pre-titered 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. 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. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
      7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      562083 Rev. 1
      Antibody Details
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      MAb11

      The MAb11 monoclonal antibody specifically binds to human tumor necrosis factor (TNF, also known as TNF-α) protein. TNF is an efficient juxtacrine, paracrine and endocrine mediator of inflammatory and immune functions. It regulates the growth and differentiation of a variety of cell types. TNF is cytotoxic for transformed cells when in conjunction with IFN-γ. It is secreted by activated monocytes/macrophages and other cells such as B cells, T cells and fibroblasts. The immunogen used to generate the MAb11 hybridoma was recombinant human TNF. The MAb11 antibody has been reported to crossreact with Rhesus Macaque TNF.

      562083 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
      562083 Rev.1
      Citations & References
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      View product citations for antibody "562083" on CiteAb

      Development References (7)

      1. Danis VA, Franic GM, Rathjen DA, Brooks PM. Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-6 on the production of immunoreactive IL-1 and TNF-alpha by human monocytes. Clin Exp Immunol. 1991; 85(1):143-150. (Clone-specific: ELISA). View Reference
      2. Jason J, Larned J. Single-cell cytokine profiles in normal humans: comparison of flow cytometric reagents and stimulation protocols. J Immunol Methods. 1997; 207(1):13-22. (Biology). 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). View Reference
      4. Rathjen DA, Cowan K, Furphy LJ, Aston R. Antigenic structure of human tumour necrosis factor: recognition of distinct regions of TNF alpha by different tumour cell receptors. Mol Immunol. 1991; 28(1-2):79-86. (Clone-specific: ELISA). View Reference
      5. Sander B, Andersson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev. 1991; 119:65-93. (Biology). View Reference
      6. Sopper S, Stahl-Hennig C, Demuth M, Johnston IC, Dorries R, ter Meulen V. Lymphocyte subsets and expression of differentiation markers in blood and lymphoid organs of rhesus monkeys. Cytometry. 1997; 29(4):351-362. (Biology). View Reference
      7. Verdier F, Aujoulat M, Condevaux F, Descotes J. Determination of lymphocyte subsets and cytokine levels in cynomolgus monkeys. Toxicology. 1995; 105(1):81-90. (Biology). View Reference
      View All (7) View Less
      562083 Rev. 1

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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.