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FITC Rat Anti-Mouse TNF
FITC Rat Anti-Mouse TNF
Expression of TNF by stimulated CD4+ and CD4- BALB/c spleen cells. After 6 h stimulation with hamster anti-mouse CD3 (Clone 145-2C11, 2 µg/ml, Cat. No. 553057) and hamster anti-mouse CD28 (Clone 37.51, 2 µg/ml, Cat. No. 553294) antibodies in the presence of BD GolgiStop™ (Cat. No. 554724; aka monensin, 3 µM) the splenocytes were stained with mouse BD FcBlock™ (1 µg/1 million cells; Cat No. 553142), then 0.06 µg of PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553049). The cells were then fixed, permeabilized, and subsequently stained with 0.06 µg of FITC-conjugated rat anti-mouse TNF antibody (FITC-MP6-XT22, Cat. No. 554418, left panel). To demonstrate specificity of staining, the binding of the FITC-MP6-XT22 antibody was blocked by preincubation of the antibody conjugate with recombinant mouse TNF (0.06 µg, Cat. No. 554589; middle panel), and by preincubation of the fixed/permeabilized cells with unlabeled MP6-XT22 antibody (2 µg, Cat. No. 554416; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and antibody blocking (right panel) specificity controls.
Expression of TNF by stimulated CD4+ and CD4- BALB/c spleen cells. After 6 h stimulation with hamster anti-mouse CD3 (Clone 145-2C11, 2 µg/ml, Cat. No. 553057) and hamster anti-mouse CD28 (Clone 37.51, 2 µg/ml, Cat. No. 553294) antibodies in the presence of BD GolgiStop™ (Cat. No. 554724; aka monensin, 3 µM) the splenocytes were stained with mouse BD FcBlock™ (1 µg/1 million cells; Cat No. 553142), then 0.06 µg of PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553049). The cells were then fixed, permeabilized, and subsequently stained with 0.06 µg of FITC-conjugated rat anti-mouse TNF antibody (FITC-MP6-XT22, Cat. No. 554418, left panel). To demonstrate specificity of staining, the binding of the FITC-MP6-XT22 antibody was blocked by preincubation of the antibody conjugate with recombinant mouse TNF (0.06 µg, Cat. No. 554589; middle panel), and by preincubation of the fixed/permeabilized cells with unlabeled MP6-XT22 antibody (2 µg, Cat. No. 554416; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and antibody blocking (right panel) specificity controls.
Product Details
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG1
Recombinant Mouse TNF
Intracellular staining (flow cytometry) (Routinely Tested)
0.5 mg/ml
AB_395379
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 FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometry: The FITC-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 ligand (e.g., recombinant mouse TNF; Cat. No 554589) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled MP6-XT22 antibody (Cat. No 554416) prior to staining. The staining technique and use of 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 cells is FITC-R3-34 (Cat. No 554684); 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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  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. Use of these products to measure activation antigens expressed on mononuclear cell subsets for the purpose of monitoring immunoregulatory status can fall under one or more claims of the following patents: US Patent Nos. 5,445,939, 5,656,446, 5,843,689; European Patent No. 319,543; Canadian Patent No. 1,296,622; Australian Patent No. 615,880; and Japanese Patent No. 2,769,156.
554418 Rev. 1
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).

This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.

554418 Rev. 1
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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FITC
Blue 488 nm
494 nm
518 nm
554418 Rev.1
Citations & References
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View product citations for antibody "554418" 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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554418 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.