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Alexa Fluor® 700 Rat anti-Mouse TNF
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Alexa  Fluor® 700  Rat anti-Mouse TNF

Expression of TNF by stimulated BALB/c spleen cells.  Splenocytes from BALB/c mice were stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and Ionomycin (500 ng, Sigma, Cat. No. I0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029).  Cells were harvested, fixed, permeabilized and stained with Alexa  Fluor® 700  Rat anti-Mouse TNF or Alexa Fluor® 700 Rat IgG1 κ Isotype Control (data not shown).  Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes.  The quadrant markers for the bivariate dot plot was based on the autofluorescence and isotype controls.

Expression of TNF by stimulated BALB/c spleen cells.  Splenocytes from BALB/c mice were stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and Ionomycin (500 ng, Sigma, Cat. No. I0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029).  Cells were harvested, fixed, permeabilized and stained with Alexa  Fluor® 700  Rat anti-Mouse TNF or Alexa Fluor® 700 Rat IgG1 κ Isotype Control (data not shown).  Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes.  The quadrant markers for the bivariate dot plot was based on the autofluorescence and isotype 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_396980
Aqueous buffered solution containing protein stabilizer and ≤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 to Alexa Fluor® 700 under optimum conditions, and unreacted Alexa Fluor® 700 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 Alexa  Fluor® 700-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 Alexa Fluor® 700 Rat IgG1 κ Isotype Control (Cat. No 558001); 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. Alexa Fluor® 700 has an adsorption maximum of ~700nm and a peak fluorescence emission of ~720nm. Before staining cells with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. 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.
  6. 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.
  7. 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.
  8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
558000 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).

558000 Rev. 1
Format Details
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Alexa Fluor™ 700
Alexa Fluor™ 700 dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 697 nm and an emission maximum (Em Max) at 719-nm. Alexa Fluor™ 700 is designed to be excited by the Red (627–640-nm) laser and detected using an optical filter centered near 720-nm (e.g., a 720/40-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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Alexa Fluor™ 700
Red 627-640 nm
697 nm
719 nm
558000 Rev.1
Citations & References
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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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558000 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.