Staining of non-transfected (left) and human CD289-transfected (right) HEK 293T cells with 1.0 µg of Rat IgG2a κ Isotype Control PE (cat. 12-4321) (open histogram) or 1.0 µg of Anti-Human CD289 (TLR9) PE (filled histogram). Total viable cells were used for analysis.
Contents: Anti-Human CD289 (TLR9) PE Catalog Number: 12-9099 Concentration: 0.2 mg/ml Formulation: Phosphate buffer pH 7.2, 150 mM NaCl, 0.09% NaN3 Storage Conditions: Store at 2-8°C. Do not freeze. Light sensitive material. Clone: eB72-1665 Host/Isotype: Rat IgG2a, κ
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12-9099-80
25 ug
12-9099-82
100 ug
Note: Several countries will continue to be supplied via distributors. Country specific prices may apply.
Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Alexa Fluor® 647 conjugated products with eFluor® 660 conjugated products.
Description
eB72-1665 is generated against a portion of human toll-like receptor 9 (aa 273-288), a molecule reported to be expressed predominantly intracellularly. TLR9 is a ~115-120 kDa molecule, which mediates response to unmethylated CpG dinucleotides in bacterial DNA. CpG DNA induces a strong T-helper-1-like inflammatory response and the proliferation of TLR9-positive human B cells. When stimulated with CpG DNA, TLR9-deficient (TLR9-/-) mice lacked splenocyte proliferation, inflammatory cytokine production from macrophages, and dendritic cell maturation, as compared with normal mice. To date, at least twelve 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. Members of the TLR family are involved in recognition and response to different microbial components including lipoproteins, peptidoglycans, and nucleic acids and play important roles in innate immunity and inflammation.
TLR9 is not detected by flow cytometry using this antibody on lysed whole human blood and/or isolated human PBMC stained for cell surface or intracellular TLR9. This may be due to limitations of antigen detection by flow cytometry. Human pDCs matured in the presence of IL-3 have been reported to stain with eB72-1665 by immunofluorescence microscopy (Nat Immunol. 5:190). Human Epithelial Cell lines were also reported to stain with this mAb (J. Immunol. 173: 1219). Further studies are needed to determine the relationship between mRNA expression and protein detection by flow cytometry.
Applications Reported
For research use only, not for diagnostic or therapeutic use. This eB72-1665 antibody has been reported for use in intracellular flow cytometric analysis.
Applications Tested
This eB72-1665 antibody has been tested by intracellular flow cytometric analysis of hTLR9 transfected cells. This can be used at less than or equal to 1 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 105 to 108 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
References
Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, Lien E, Nilsen NJ, Espevik T, Golenbock DT. 2004. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol. 5(2):190-8.
Juliane Platz, Christoph Beisswenger, Alexander Dalpke, Rembert Koczulla, Olaf Pinkenburg, Claus Vogelmeier, and Robert Bals. 2004. Microbial DNA Induces a Host Defense Reaction of Human Respiratory Epithelial Cells. J. Immunol. 173: 1219 - 1223.
Jozsef L, Khreiss T, El Kebir D, Filep JG.
Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils.
J Immunol. 2006 Jan 15;176(2):1195-202. (eB72-1665, FC, PubMed)
Schroeder JT, Bieneman AP, et al. 2005. TLR9- and FcepsilonRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression. J Immunol. 175(9):5724-31. (eB72-1665, WB, PubMed)
Leifer CA, Kennedy MN, et al. 2004. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol. 173(2):1179-83. (Immunofluorescence and IP, PubMed)
Dasari P, Nicholson IC, et al. 2005. Expression of toll-like receptors on B lymphocytes. Cell Immunol. 236(1-2):140-5. (eB72-1665, IC flow, PubMed)
Peduzzi E, Groeper C, et al. 2007. Local activation of the innate immune system in Buruli ulcer lesions. J Invest Dermatol. 127(3):638-45. (eB72-1665, IHC paraffin, PubMed)
