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BB700 Rat Anti-Mouse TNF
BB700 Rat Anti-Mouse TNF
Two color flow cytometric analysis of TNF expression by stimulated mouse splenocytes. Mouse splenic leucocytes were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139; 50 ng/ml) and Ionomycin (Sigma I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with stain buffer, and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722), and then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with FITC Rat Anti-Mouse CD4 antibody (Cat. No. 553047/553046/561835) and either BD Horizon™ BB700 Rat IgG1, κ Isotype Control (Cat. No. 566420; Left Plot) or BD Horizon BB700 Rat Anti-Mouse TNF antibody (Cat. No. 566510/566511; Right Plot) at 0.5 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. Flow cytometric dot plots showing the correlated expression of TNF (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated leucocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System. Data shown on this Technical Data Sheet are not lot specific.
Two color flow cytometric analysis of TNF expression by stimulated mouse splenocytes. Mouse splenic leucocytes were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139; 50 ng/ml) and Ionomycin (Sigma I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with stain buffer, and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722), and then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with FITC Rat Anti-Mouse CD4 antibody (Cat. No. 553047/553046/561835) and either BD Horizon™ BB700 Rat IgG1, κ Isotype Control (Cat. No. 566420; Left Plot) or BD Horizon BB700 Rat Anti-Mouse TNF antibody (Cat. No. 566510/566511; Right Plot) at 0.5 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. Flow cytometric dot plots showing the correlated expression of TNF (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated leucocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System. Data shown on this Technical Data Sheet are not lot specific.
Product Details
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BD Horizon™
Tnf; Tnfa; TNF alpha; TNF-a; Tnfsf1a; Tnfsf2; TNFSF2; Cachectin; DIF
Mouse (QC Testing)
Rat IgG1
Recombinant Mouse TNF
Flow cytometry (Routinely Tested)
0.2 mg/ml
AB_2869775
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 with BD Horizon™ BB700 under optimum conditions, and unconjugated antibody and free BD Horizon BB700 were removed.

Recommended Assay Procedures

     For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions.  More information can be found in the Technical Data Sheet for the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).

     When setting up compensation, it is recommended to compare spillover values obtained from cells and BD™ CompBeads to ensure that beads will provide sufficiently accurate spillover values.

     For optimal results, it is recommended to perform two washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescent staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.

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. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. Diagnostic uses require a separate license.
  6. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  7. BD Horizon Brilliant Blue 700 is covered by one or more of the following US patents: 8,455,613 and 8,575,303.
  8. Cy is a trademark of GE Healthcare.
  9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
566511 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).

The antibody was conjugated to BD Horizon BB700, which is part of the BD Horizon Brilliant™ Blue family of dyes.   It is a polymer-based tandem dye developed exclusively by BD Biosciences.  With an excitation max of 485 nm and an emission max of 693 nm, BD Horizon BB700 can be excited by the 488 nm laser and detected in a standard PerCP-Cy™5.5 set (eg, 695/40-nm filter). This dye provides a much brighter alternative to PerCP-Cy5.5 with less cross laser excitation off the 405 nm and 355 nm lasers.

566511 Rev. 1
Format Details
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BB700
The BD Horizon Brilliant™ Blue 700 (BB700) Dye is part of the BD Horizon Brilliant™ Blue family of dyes. This tandem fluorochrome is comprised of a polymer-technology dye donor with an excitation maximum (Ex Max) of 476-nm and an acceptor dye with an emission maximum (Em Max) at 695-nm. Driven by BD innovation, BB700 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 695-nm (e.g., a 695/20-nm bandpass filter). The donor dye can be excited by the Violet (405 nm) laser and the acceptor dye can be excited by the red (627–640 nm) laser resulting in cross-laser excitation and fluorescence spillover. BB700 Reagents are significantly brighter than equivalent PerCP or PerCP-Cy5.5 reagents and are less sensitive to photobleaching. In addition, BB700 shows much less excitation by the violet (407-nm) laser resulting in less spillover. BB700 has minimal yellow green (562-nm) excitation and is ideal for instruments with both blue (488-nm) and yellow green (562-nm) lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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BB700
Blue 488 nm
476 nm
695 nm
566511 Rev.1
Citations & References
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Development References (7)

  1. Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
  2. 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: Blocking, ELISA). View Reference
  3. Bitsaktsis C, Winslow G. Fatal recall responses mediated by CD8 T cells during intracellular bacterial challenge infection. J Immunol. 2006; 177(7):4644-4651. (Clone-specific: Blocking). View Reference
  4. 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: Immunohistochemistry). View Reference
  5. 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: Immunohistochemistry). View Reference
  6. 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, IC/FCM Block). View Reference
  7. Yang J, Kawamura I, Zhu H, Mitsuyama M. Involvement of natural killer cells in nitric oxide production by spleen cells after stimulation with Mycobacterium bovis BCG. Study of the mechanism of the different abilities of viable and killed BCG. J Immunol. 1995; 155(12):5728-5735. (Clone-specific: Blocking). View Reference
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566511 Rev. 1

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.