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Flow cytometric analysis for TNF in stimulated Rhesus macaque peripheral blood mononuclear cells (PBMC). PBMC from Rhesus macaque were stimulated for 6 hours with 50 ng/mL PMA (Sigma-Aldrich Cat. No. P-8139) and 500 ng/mL calcium ionophore A23187 (Sigma-Aldrich Cat. No. C-9275) in the presence of BD GolgiStop™ (Cat. No. 554724). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm™ (Cat. No. 554714) followed by staining with either a PE-Cy™7 Mouse IgG1, κ isotype control (Cat. No. 557646; left panel) or with the PE-Cy™7 Mouse Anti-Human TNF antibody (Cat. No. 560678/557647; right panel). Dot plots were derived from gated events based on light scattering characteristics for lymphocytes. Flow cytometry was performed on a BD™ LSR II flow cytometry system.
BD Pharmingen™ PE-Cy™7 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
Flow cytometry: The MAb11 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate TNF producing cells within mixed cell populations. A useful control investigators may consider using for demonstrating specificity of staining, is to pre-block with one of the following reagents: (1) recombinant human TNF (Cat. No. 554618) or (2) unlabeled MAb11 antibody (Cat. No. 554510), prior to staining.
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.
- Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
- PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of GE Healthcare.
- 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.
MAb11 has been predicted to be reactive on other non-human primate samples (e.g Baboon, Cynomolgus). Investigators are advised that the PE-Cy™7 Mouse Anti-Human TNF (clone MAb11) antibody is not routinely tested on these other non-human primate samples.
Development References (14)
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Petyovka N, Lyach L, Voitenok NN. Homologous ELISA for detection of oligomeric human TNF: properties of the assay. J Immunol Methods. 1995; 186(2):161-170. (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. (Biology). View Reference
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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.