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PE Rat Anti-Mouse TNF
PE Rat Anti-Mouse TNF
Expression of TNF by MiCK-1 cells. MiCK-1 cells (Cat. No. 554652) were stained with 0.06 ug of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.06 ug of PE-conjugated rat anti-mouse TNF antibody (PE-MP6-XT22, Cat. No. 554419) by using the Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP6-XT22 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse TNF (0.25 µg, Cat. No. 554589; right panel), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled MP6-XT22 mAb (2 µg, Cat. No. 554416; data not shown). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (right panel) and unlabelled antibody blocking specificity controls.  
Expression of TNF by MiCK-1 cells. MiCK-1 cells (Cat. No. 554652) were stained with 0.06 ug of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.06 ug of PE-conjugated rat anti-mouse TNF antibody (PE-MP6-XT22, Cat. No. 554419) by using the Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP6-XT22 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse TNF (0.25 µg, Cat. No. 554589; right panel), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled MP6-XT22 mAb (2 µg, Cat. No. 554416; data not shown). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (right panel) and unlabelled antibody blocking specificity controls.  
Product Details
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG1
Recombinant Mouse TNF
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_395380
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.

Recommended Assay Procedures

Recommended Assay Procedure:

The PE-conjugated MP6-XT22 antibody can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate TNF-producing cells within mixed cell populations (see figure). 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 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 MP6-XT22 antibody with a molar excess of ligand (e.g., recombinant mouse TNF; Cat No. 554589) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabelled MP6-XT22 antibody (Cat. No. 554416) prior to staining. The staining technique and 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 and human cells is PE-R3-34 (Cat. No. 554685); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/ 1 million cells).

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.
554419 Rev. 2
Antibody Details
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MP6-XT22

The MP6-XT22 antibody specifically binds to mouse Tumor Necrosis Factor (TNF, also known as TNF-α).  TNF is produced by many activated cell types including monocytes, macrophages, astrocytes, granulocytes, mast cells, T and B lymphocytes, NK cells, keratinocytes, fibroblasts, adipocytes, and certain tumor cells. Activated cells express type II transmembrane TNF glycoproteins that associate as homotrimeric complexes. After enzymatic cleavage, the extracellular regions of membrane TNF are shed as soluble homotrimers. TNF is a potent multifunctional cytokine that can exert regulatory and cytotoxic effects on a wide range of normal lymphoid and non-lymphoid cells and tumor cells. Although TNF serves as a primary mediator in protective immune responses against microbial and viral pathogens, it can also drive systemic pathophysiologic responses including septic shock, cachexia and autoimmune diseases. Mouse TNF exerts its biological activities by binding and signaling through cell surface membrane Type I and Type II TNF Receptors (aka, TNFRI/CD120a and TNFRII/CD120b, respectively).

554419 Rev. 2
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
554419 Rev.2
Citations & References
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View product citations for antibody "554419" on CiteAb

Development References (4)

  1. 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). View Reference
  2. Hunter CA, Litton MJ, Remington JS, Abrams JS. Immunocytochemical detection of cytokines in the lymph nodes and brains of mice resistant or susceptible to toxoplasmic encephalitis. J Infect Dis. 1994; 170(4):939-945. (Clone-specific). View Reference
  3. Litton MJ, Sander B, Murphy E, O'Garra A, Abrams JS. Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J Immunol Methods. 1994; 175(1):47-58. (Clone-specific). View Reference
  4. 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). View Reference
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554419 Rev. 2

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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.