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APC Mouse Anti-Human TNF
APC Mouse Anti-Human TNF

Expression of TNF by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (Sigma, Cat. #C-9275) in the presence of GolgiStop™ (Cat. No. 554724; aka monensin 2 µM). The PBMC were stained with FITC Mouse Anti-Human CD3 (Cat. No. 555332), fixed, permeabilized, and subsequently stained with 0.25 µg of APC Mouse Anti-Human TNF (Cat. No. 554514; left panel). To demonstrate specificity of staining, the binding of APC Mouse Anti-Human TNF was blocked by the preincubation of the conjugated antibody with recombinant human TNF (0.5 µg; Cat. No. 554618; see middle panel), and by preincubation of the fixed/permeabilized cells with the Purified Mouse Anti-Human TNF antibody (10 µg, Cat. No. 554510; right panel) prior to staining with the APC Mouse Anti-Human TNF. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine-blocking and unlabeled antibody-blocking specificity controls. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNE or red diode laser. These include the dual laser FACStarPLUS™, FACS Vantage™ or FACSCalibur™.

Expression of TNF by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (Sigma, Cat. #C-9275) in the presence of GolgiStop™ (Cat. No. 554724; aka monensin 2 µM). The PBMC were stained with FITC Mouse Anti-Human CD3 (Cat. No. 555332), fixed, permeabilized, and subsequently stained with 0.25 µg of APC Mouse Anti-Human TNF (Cat. No. 554514; left panel). To demonstrate specificity of staining, the binding of APC Mouse Anti-Human TNF was blocked by the preincubation of the conjugated antibody with recombinant human TNF (0.5 µg; Cat. No. 554618; see middle panel), and by preincubation of the fixed/permeabilized cells with the Purified Mouse Anti-Human TNF antibody (10 µg, Cat. No. 554510; right panel) prior to staining with the APC Mouse Anti-Human TNF. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine-blocking and unlabeled antibody-blocking specificity controls. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNE or red diode laser. These include the dual laser FACStarPLUS™, FACS Vantage™ or FACSCalibur™.

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)
0.2 mg/ml
AB_398566
Aqueous buffered solution containing ≤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 to APC under optimum conditions, and unconjugated antibody and free APC were removed.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometry: The APC-conjugated MAb11 antibody can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate TNF-producing cells within mixed cell populations (see image). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/million cells). For specific methodology, please visit the protocols section under "Cytokines (Intracellular Staining)" or "Intracellular Flow" at our website, http://www.bdbiosciences.com/us/s/resources.

A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated MAb11 antibody with a ligand (e.g., recombinant human TNF; Cat No. 554618) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled MAb11 antibody (Cat. No. 554510) prior to staining. The staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is APC-MOPC-21 (Cat. No. 554681); use at comparable concentrations to antibody of interest.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. 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.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  6. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
554514 Rev. 3
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.

554514 Rev. 3
Format Details
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APC
Allophycocyanin (APC), is part of the BD family of phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 651 nm and an emission maximum (Em Max) at 660 nm. APC is designed to be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 660 nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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APC
Red 627-640 nm
651 nm
660 nm
554514 Rev.3
Citations & References
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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
554514 Rev. 3

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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.