Staining of thioglycolate-induced peritoneal exudate cells with 0.5 µg of Rat IgG2a K Isotype Control PE-Cyanine7 (Product # 25-4321) (open histogram) or 0.5 µg of Anti-Mouse TLR4/MD-2 Complex PE-Cyanine7 (filled histogram). Total viable cells were used for analysis.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Rat / IgG2a, kappa|
|Storage buffer||PBS, pH 7.2, with 0.1% gelatin|
|Contains||0.09% sodium azide|
|Storage Conditions||4° C, store in dark, DO NOT FREEZE!|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||2 µg/test|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Description: The MTS510 monoclonal antibody reacts with the mouse Toll-like receptor 4 (TLR4)/MD-2 complex. At least ten members of the Toll family have been identified. This family of type I transmembrane proteins is characterized by an extracellular domain with leucine-rich repeats and a cytoplasmic domain with homology to the type I IL-1 receptor. Two of these receptors, TLR2 and TLR4, are pattern recognition receptors and signaling molecules in response to bacterial lipoproteins and have been implicated in innate immunity and inflammation. TLR4 physically associates with MD-2, and together with CD14, this complex is responsible for LPS recognition and signaling. In the mouse, TLR4 is expressed by thioglycolate-elicited peritoneal macrophages. Incubation of peritoneal macrophages with LPS results in down regulation of surface TLR4/MD-2. The TLR4 gene is defective in C3H/HeJ and C57BL/10ScCr mice, both of which have been well characterized as hyporesponders to LPS.
The MTS510 monoclonal antibody co-immunoprecipitates MD-2 (~30 kDa) and TLR4 (~100 kDa), and preferentially reacts with TLR4 that is associated with MD-2. In comparison, binding of the UT41 monoclonal antibody occurs with and without formation of the TLR4/MD-2 complex. Please contact eBioscience Technical Support for further information.
Applications Reported: This MTS510 antibody has been reported for use in flow cytometric analysis.
Applications Tested: This MTS510 antibody has been tested by flow cytometric analysis of mouse peritoneal-exudate cells. This can be used at less than or equal to 2 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
Light sensitivity: This tandem dye is sensitive photo-induced oxidation. Please protect this vial and stained samples from light.
Fixation: Samples can be stored in IC Fixation Buffer (cat. 00-8222) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Excitation: 488-561 nm; Emission: 775 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser
MD-2 is a member of the Toll/interleukin-1 receptor (TIR) family, a group of proteins that include the Toll-like receptors (TLRs). TLRs are signaling molecules that recognize different pathogen-associated molecular patterns (PAMPs) and serve as an important link between the innate and adaptive immune responses. TLR4, the major signaling receptor for lipopolysaccharide (LPS), requires the binding of MD-2 to its extracellular region for maximal response to LPS. The specificity of this response is determined by the species of MD-2; e.g., human MD-2 transfected into mouse cells can cause mouse TLR4 to react to LPS analogs that are normally antagonistic to human but not mouse TLR4.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||TLRs regulate the gatekeeping functions of the intestinal follicle-associated epithelium.||Chabot S,Wagner JS,Farrant S,Neutra MR||Journal of immunology (Baltimore, Md. : 1950) (176:4275)||2006|
|Not Applicable||Not Cited||Strategic compartmentalization of Toll-like receptor 4 in the mouse gut.||Ortega-Cava CF,Ishihara S,Rumi MA,Kawashima K,Ishimura N,Kazumori H,Udagawa J,Kadowaki Y,Kinoshita Y||Journal of immunology (Baltimore, Md. : 1950) (170:3977)||2003|
|Not Applicable||Not Cited||Acetylsalicylic acid-induced release of HSP70 from mast cells results in cell activation through TLR pathway.||Mortaz E,Redegeld FA,Nijkamp FP,Wong HR,Engels F||Experimental hematology (34:8)||2006|
|Not Applicable||Not Cited||Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages.||Qi HY,Shelhamer JH||The Journal of biological chemistry (280:38969)||2005|
|Not Applicable||Not Cited||Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways.||Sato S,Nomura F,Kawai T,Takeuchi O,Mühlradt PF,Takeda K,Akira S||Journal of immunology (Baltimore, Md. : 1950) (165:7096)||2000|
|Not Applicable||Not Cited||Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages.||Akashi S,Shimazu R,Ogata H,Nagai Y,Takeda K,Kimoto M,Miyake K||Journal of immunology (Baltimore, Md. : 1950) (164:3471)||2000|