Description
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).
Recommended Assay Procedure: The Alexa Fluor® 488-conjugated MP6-XT22 antibody (Cat No. 557719) is 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 the chapter on intracellular staining for flow cytometric analysis 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. 554417) 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® 488 conjugated R3-34 (Cat. No. 557720); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/ 1 million cells).
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
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.
