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BV750 Rat Anti-Mouse TNF
BV750 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 APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and either BD Horizon™ BV750 Rat IgG1, κ Isotype Control (Cat. No. 566367; Left Plot) or BD Horizon BV750 Rat Anti-Mouse TNF antibody (Cat. No. 566365; Right Plot) at 0.25 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 APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and either BD Horizon™ BV750 Rat IgG1, κ Isotype Control (Cat. No. 566367; Left Plot) or BD Horizon BV750 Rat Anti-Mouse TNF antibody (Cat. No. 566365; Right Plot) at 0.25 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
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_2739712
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 BV750 under optimum conditions, and unconjugated antibody and free BD Horizon BV750 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 of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349).

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. 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.
  6. BD Horizon Brilliant™ Violet 750 is covered by one or more of the following US patents: 8,158,444; 8,802,450; 8,575,303; 8,455,613; 8,227,187; 8,841,072; 8,110,673.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
566365 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 BV750 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 750-nm. BD Horizon Brilliant BV750 can be excited by the violet laser (405 nm) and detected with a 750/30 nm filter with a 740 nm long pass. Due to spectral differences between labeled cells and beads, using BD™ CompBeads can result in incorrect spillover values when used with BD Horizon BV750 reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended.

566365 Rev. 1
Format Details
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BV750
The BD Horizon Brilliant Violet™ 750 (BV750) dye is part of the BD Horizon Brilliant Violet™ family of dyes. This tandem fluorochrome is comprised of a BV421 donor with an excitation maximum (Ex Max) of 409-nm and an acceptor dye with an emission maximum (Em Max) at 754-nm. BV750, driven by BD innovation, is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 750-nm (e.g., a 750/30 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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BV750
Violet 405 nm
409 nm
754 nm
566365 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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566365 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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.