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Alexa Fluor® 647 Rat Anti-Mouse TNF
Alexa Fluor® 647 Rat Anti-Mouse TNF

Expression of TNF by stimulated CD4+ and CD4-BALB/c spleen cells. Splenocytes from BALB/C mice were stimulated for 4 hrs with PMA (5 ng/ml, Sigma) and Ionomycin (500 ng, Sigma) in the presence of BD GolgiPlug™ (Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553048) and either rat anti-mouse TNF antibody (Alexa 647-MP6-XT22, Cat. No. 557730), (left panel) or immunoglobulin isotype control (Alexa 647-R3-34, Cat. No. 557731), (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa 647-MP6-XT22 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse TNF (0.25 µg, Cat. No. 554589, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled MP6-XT22 antibody (5 µg, Cat. No. 554416, data not shown) prior to stainining. Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes. The quadarant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.

Expression of TNF by stimulated CD4+ and CD4-BALB/c spleen cells. Splenocytes from BALB/C mice were stimulated for 4 hrs with PMA (5 ng/ml, Sigma) and Ionomycin (500 ng, Sigma) in the presence of BD GolgiPlug™ (Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553048) and either rat anti-mouse TNF antibody (Alexa 647-MP6-XT22, Cat. No. 557730), (left panel) or immunoglobulin isotype control (Alexa 647-R3-34, Cat. No. 557731), (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa 647-MP6-XT22 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse TNF (0.25 µg, Cat. No. 554589, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled MP6-XT22 antibody (5 µg, Cat. No. 554416, data not shown) prior to stainining. Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes. The quadarant markers for the bivariate dot plots were set 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_396838
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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Recommended Assay Procedure:

The FITC-, PE-, APC-, and PE-Cy7-, Alexa Fluor® 488- and Alexa Fluor® 647-conjugated MP6-XT22 antibody (Cat No. 554418, 554419, 554420, 557644, 557719 and 557730) are available for multicolor immunofluorescent staining and flow cytometric analysis 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 the protocols section or chapter on intracellular flow cytometry in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.

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 Alexa Fluor® 647-R3-34 (Cat. No. 557731). Use isotype controls 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. 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.
  5. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  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. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
557730 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).

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.

557730 Rev. 2
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
557730 Rev.2
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
View All (4) View Less
557730 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.