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Expression of TNF by stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and Ionomycin (500 ng, Sigma, P-8139) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with PE-Cy™7 Mouse Anti-Human CD8 (Cat. No. 557746) and either Alexa Fluor® 488 Mouse Anti-Human TNF (Cat. No. 557722; left panel) or Alexa Fluor® 488 Mouse IgG1 κ Isotype Control (Cat. No. 557721; right panel). To demonstrate specificity of staining the binding of Alexa Fluor® 488 Mouse Anti-Human TNF was blocked by the preincubation of the conjugated antibody with molar excess of recombinant human TNF (0.25 µg, Cat. No. 554618, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of Purified Mouse Anti-Human TNF (5 µg, Cat. No. 554510, data not shown) prior to staining. Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
BD Pharmingen™ Alexa Fluor® 488 Mouse Anti-Human TNF
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
Recommended Assay Procedures
Immunofluorescent Staining and Flow Cytometric Analysis: The MAb11 antibody is useful for intracellular immunofluorescent staining and flow cytometric analysis to identify and enumerate TNF producing cells within mixed cell populations. The Alexa Fluor® 488-conjugated MAb11 antibody (Cat. No. 557722) is especially suitable for these studies (see image). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be used at 5 µl/test. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit our web site, http://www.bdbiosciences.com/us/s/resources, and go to the protocols section under "Cytokines (Intracellular Staining" or "Intracellular Flow".
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).
- An isotype control should be used at the same concentration as the antibody of interest.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
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.
Development References (7)
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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
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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
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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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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
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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
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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
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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
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.