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Datasheet

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Monoclonal Antibody to TLR9/CD289 (Clone 26C593.2)
Cat.No
IMG-305D
Description
TLR9/CD289
Unit
0.1 mg
Price(USD)
345.00
MSDS
View MSDS
Catalog No : IMG-305D
Contents : 0.1 mg antibody in 0.2 ml PBS containing 0.05% sodium azide. Sodium azide is highly toxic.
Isotype : Mouse IgG1, Kappa
Clone : 26C593.2
Purification : Protein G Chromatography
Species : Dog, Horse, Human, Mouse, Rat, Rhesus Monkey
Host : Mouse
Application
Confocal Microscopy: see Greene et. al. (2005)
FC (Intracellular): 0.1-0.5 ug/1x10^6 cells
Flow (Cell Surface): see Lee et al. (2006)
IHC (frozen): 10-20 ug/ml (see Miller et al, 2005).
IHC (paraffin): see Martin-Armas et al. (2005)
Storage
Product is light- and temperature-sensitive.  Store in the dark at 4°C.  Do not freeze.
Recommended Positive Control : Ramos, human PBMCs 

Background
The Toll-like receptor (TLR) family in mammal comprises a family of transmembrane proteins characterized by multiple copies of leucine rich repeats in the extracellular domain and IL-1 receptor motif in the cytoplasmic domain. Like its counterparts in Drosophila, TLRs signal through adaptor molecules (1). The TLR family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition (2). Ten human homologs of TLRs (TLR1-10) have been described (3). By using a BLAST search, Hemmi et al., 2000 (4) have identified and subsequently isolated a cDNA coding for TLR9. Gene knockout experiments suggest that TLR9 acts as a receptor for unmethylated CpG dinucleotides in the bacterial DNA (4). Human and mouse TLR9 share an overall amino-acid identity of 75.5%. TLR9 is highly expressed in spleen.

Antigen
This antibody was developed against a KLH-conjugated synthetic peptide corresponding to amino acids 268-284 (CPRHFPQLHPDTFSHLS) of human TLR9.

Genebank Info (Protein)
NP_059138

 

Intracellular flow analysis of TLR9 in human PBMCs using 0.2 ug of IMG-305D.  Shaded histogram represents cells without antibody; green represents a mouse IgG1-PE isotype control (BD Biosciences, Cat no. 554680); red represents anti-TLR9 antibody.

Intracellular flow analysis of TLR9 in Ramos cells using 0.1 ug of IMG-305D.  Shaded histogram represents Ramos cells without antibody; green represents a mouse IgG1-PE isotype control (BD Biosciences, Cat no. 554680); red represents anti-TLR9 antibody.

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3. 10083K [IC-Flow (Intracellular Staining Flow Assay) Kit]
4. IMG-2209H [CpG ODN (2006) with negative control oligo, TLR9 ligand (human)]

Reference
1. Muzio M, Natoli G, Saccani S, Levrero M, and Mantovani A. J. Exp. Med. 187: 2097-2101 (1998).
2. Medzhitov R and Janeway CA. Cell 91: 295-298 (1997).
3. Chuang TH and Ulevitch RJ. Biochim. Biophys. Acta 1518 (1-2):157-161 (2001)
4. Takeuchi O, Kawai T, Sanjo H, Copeland NG, Gilbert DJ, Jenkins NA, Takeda K, and Akira S. Gene 231: 59-65 (1999).
5. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, and Shizo A. Nature 408: 740-745 (2000).


Product Citations
1. Cytoplasmic domain-mediated dimerizations of toll-like receptor 4 observed by lactamase enzyme fragment complementation. Hyun-Ku Lee, Stefan Dunzendorfer, and Peter S. Tobias.  . J. Biol. Chem., Dec 2003; 10.1074/jbc.M311564200. Imgenex antibodies cited: TLR9 (IMG-305) [WB, Fig5B (HEK293 cells)].
2. Evidence of Toll-like receptor molecules on human platelets. Fabrice Cognasse, Hind Hamzeh, Patricia Chavarin, Sophie Acquart, Christian Genin and Olivier Garraud. Immunology and Cell Biology. Volume 83 Issue 2 Page 196 - April 2005. doi:10.1111/j.1440-1711.2005.01314. The following TLR antibodies were cited in this publication: TLR2-PE (IMG-416D), TLR4-PE (IMG-417D), TLR6 (IMG-304A), TLR8-PE (IMG-321D), TLR9-PE (IMG-305D). Applications: Intracellular Flow Cytomety and Cell Surface Flow Cytometry: Figs 1 and 2. A comparison of staining results, intracellular versus cell surface flow cytometry is shown. Cell type: Human platelets
3. TGF-a Regulates TLR Expression and Function on Epidermal Keratinocytes. Lloyd S. Miller, Ole E. Sřrensen, Philip T. Liu, H. Ray Jalian, Deborah Eshtiaghpour, Behnaz E. Behmanesh, Woosin Chung, Timothy D. Starner, Jenny Kim,, Peter A. Sieling, Tomas Ganz and Robert L. Modlin. J. Immunol, 174: 6137-6143 (2005). Imgenex antibodies cited: 1. TLR5 (IMG-664) [IHC-frozen, Fig 1 (normal human skin and psoriasis, primary human organotypic keratinocyte culture)].  2. TLR9 (IMG-305A) [IHC-frozen, Fig 1 (normal human skin and psoriasis, primary human organotypic keratinocyte culture)]. 
4. Intracellular Signaling Mechanisms Regulating Toll-Like Receptor-Mediated Activation of Eosinophils.  Wong CK, PFY Cheung, WK IP and CWK Lam. Am. J. Respir. Cell Mol. Biol.doi:10.1165/rcmb.2006-0457OC (2007), in press.   Imgenex antibodies cited (human blood eosinophils and neutrophils from buffy coat): For WB, Fig. 1A: TLR1 (IMG-5012),  TLR5 (IMG-664), TLR6 (IMG-304A), TLR7 (IMG-540), TLR8 (IMG-321A), TLR9 (IMG-305A). For Flow (Intracellular) and Flow (Surface), Fig. 1B: TLR1 (IMG-5021), TLR2 (IMG-416C), TLR3 (IMG-315C), TLR4 (IMG-417C), TLR5 (IMG-663C), TLR6 (IMG-304C), TLR7 (IMG-665A), TLR8 (IMG-321C), TLR9 (IMG-305C).
5. Activation of anti-hepatitis C virus responses via Toll-like receptor 7. Lee J, CCN Wu, KJ Lee T-H Chuang, K Katakura, Y-T Liu, M Chan, R Tawatao, M Chung, C Shen, HB Cottam, MMC Lai, E Raz and DA Carson. PNAS 103:1828-1833 (2006). Imgenex antibodies cited in this study:  Flow (Intracellular) and Flow (Cell Surface) in human Huh-7 hepatoma cells, Fig. 1A [TLR7 (IMG-665A], [TLR8 (IMG-321C)], and [TLR9 (IMG-305D)];  IHC (paraffin) on Huh-7 cells, normal fibroblasts, HCV-infected liver, and liver carcinoma, Fig 1C-E (TLR7, IMG-665A).  The specificity of the TLR7 antibody (IMG-665A) was validated using TLR7 293 transfected cells in Flow (Intracellular) (Supplemental Data, Fig. 5).  
6. Surface expression of Toll-like receptor 9 is upregulated on intestinal epithelial cells in response to pathogenic bacterial DNA. Ewaschuk JB, JL Backer, TA Churchill, F Obermeier, DO Krause, and KL Masden. Infection and Immunity 75:2572-2579 (2007). Imgenex antibody cited: IMG-305A in human HT-29 colon carcinoma cells Flow (Intracellular) Fig 4, IF (Fig 5), WB (Fig 6) and in mouse colon tissue (from normal and germ-free mice) for WB (Fig 7A, B) and IHC (paraffin) (Fig 7C).     
7. Toll-like receptor 9 expression in murine and human adrenal glands and possible implications during inflammation. Tran N, A Koch, R Berkels, O Boehm, PA Zacharowski, G Baumgarten, P Knuefermann, M Schott, W Kanczkowski, SR Bornstein, SL LIghtman, and K Zacharowski. J Clin Endocrin Metab. Doi:10.1210/jc.2006-2697 In press (2007). Imgenex antibodies cited (mouse spleen and adrenal tissue, mouse RAW264.7 macrophage cell line, human adrenal tissue: TLR9 [(IMG-305A) IHC (paraffin): Figs 1C-D, F and 6; IF/ICC: Figs 1E and 5]; TLR9 [(IMG-431) WB: Figs 1B, 3C, and 5G].  The TLR9 IMG-431 antibody was validated by WB using adrenal gland tissue from TLR9-/- and wt mice (Fig 1B).    
8. Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct cytokine gene expression patterns in Rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing Rhesus monkey plasmacytoid dendritic cells. Abel K, Y Wang, L Fritts, E Sanchez, E Chung, P Fitzgerald-Bocarsly, AM Krieg, and CJ Miller. Clinical and Diagnostic Laboratory Immunology 12:606-621 (2005).  Imgenex antibodies cited: IMG-305C [Flow (Intracellular), Fig. 5] on Rhesus monkey spleen cell suspensions and PBMC.
9. Toll-like receptors in cellular subsets of human tonsil T cells: altered expression during recurrent tonsillitis. Mansson A, M Adner and LO Cardell. Respiratory Resesarch doi:10.1186/1465-9921-7-36. (2006).  Imgenex antibodies cited (human tonsils separated into cell subtypes): 1. TLR3 [IMG-315D (Flow-Intracellular), Figs 5 and 6]. 2. TLR5 [IMG-663A (Flow-Intracellular), Fig 6]. 3. TLR9 [IMG-305C (Flow-Intracellular), Fig 4.].
10. TLR-Induced Inflammation in Cystic Fibrosis and Non-Cystic Fibrosis Airway Epithelial Cells. Catherine M. Greene, Tomás P. Carroll, Stephen G. J. Smith, Clifford C. Taggart, James Devaney, Siobhan Griffin, Shane J. O’Neill, and Noel G. McElvaney. J. Immunol., 174: 1638-1646 (2005). Imgenex antibodies cited: 1. TLR9 (IMG-305) [Confocal Microscopy, Fig.1A and 1B (CFTE29o- and 16HBE14o- cells)].
11. Maintenance of colonic homeostasis by distinctive apical TLR9 signaling in intestinal epithelial cells. Lee J., J-H Mo, K. Katakura, I. Alkalay, A. N. Rucker, Y-T. Liu, H-K. Lee, C. Shen, G. Cojocaru, S. Shenouda, M. Kagnoff, L. Eckmann, Y. Ben-Neriah, and E. Raz., Nature Cell Biol., 8,doi:10.1038/ncb1500 (2006). Imgenex antibodies cited: 1. TLR9 (IMG-305) [Confocal Microscopy, Fig.2 (intestinal epithelial cells)]. [Flow (cell surface), Fig.S1 (293-HEK TLR9 transfected cells) and Fig.S2 (Caco-2 intestinal epithelial cells)], TLR9 mAb was transfected validated by flow (cell surface) in Fig. S1. [WB and IP, Fig S1 (293-HEK TLR9 transfected cells and Caco-2 intestinal epithelial cells), TLR9 mAb was transfected validated by IP/WB.

12. Role of pathogenic auto-antibody production by Toll-like receptor 9 of B cells in active systemic lupus erythematosus. Nakano S, S Morimoto, J Suzuki, K Nozawa, H Amano, Y Tokano, Y Takasaki. Rheumatology 47: 145-149 (2008). Imgenex antibody cited: IMG-305C (TLR9-FITC) for Flow (Intracellular),  human peripheral blood B cells from normal and SLE patients, Figs 1C, 2, 3.
13. Induction of pro-inflammatory programs in enteroendocrine cells by the Toll-like receptor agonists flagellin and bacterial LPS. Selleri S, Palazzo M, Deola S, Wang E, Balsari A, Marincola F, Rumio C.  International Immunology doi:10.1093/intimm/dxn055 (2008). TLR9-FITC (IMG-305C): Flow (intracellular), Human colon neuroendocrine LCC-18 cell line: Fig. 4d.
14. Expression of TLR9 within human glioblastoma. Meng Y, M Kujas, Y Marie, S Paris, J Thillet, J Delattre, A Carpentier. Journal of Neuro-Oncology: 10.1007/s11060-008-9536-2 (2008). IMG-305A: human glioma cell lines SNB19, HTB13 and U87, IHC(parrafin): Fig. 2a; IF: Fig. 2c,f.
15. TLR9 is expressed in idiopathic interstitial pneumonia and its activation promotes in vitro myofibroblast differentiation. Meneghin A, E Choi, H Evanoff, S Kunkel, F martinez, K Flaherty, G Toews, C Hogaboam. Histochemisty and Cell Biology: 10.1007/s00418-0080466-z (2008). Imgenex products cited (human lung fibroblast cell lines):
1. IMG-305A (TLR9): IHC (paraffin), Fig. 2a,b,c,d,e,f; 3b,d; 4a,b,c,d,e,rf,g,h; 5b,d,f; 6b,d,f,h.
2. IMG-305B (TLR9 biotin): IF, Fig. 8a,b,c,d
.

16.

STAT3 and STAT5-dependent pathways competitively regulate the pan-differentiation of DC34pos cells into tumor-competent dendritic cells. Cohen P, Koski G, Czerniecki B, Bunting K, Fu X, Wang Z, Zhang W, Carter C, Awad M, Distel C, Nagem H, Paustian C, Johnson T, Tisdale J, Shu S. Immunobiology 112: 1832-1843 (2008). Imgenex antibodies cited [Flow (intracellular), mouse bone marrow cells, Supplementary Fig. S4]:
1. TLR3 FITC (IMG-315C)
2. TLR4 FITC (IMG-5031C)
3. TLR7 (IMG-665A)
4. TLR8 FITC (IMG-321C)
5. TLR9 FITC (IMG-305C)

17. CD300a/c regulate type 1 intergeron and TNF-α secretion by human plasmacytoid dendritic cells stimulated with TLR7 and TLR9 ligands. Ju X, M Zenke, D Hart, G Clark. Blood 112: 1184-1194 (2008). IMG-305D: Flow (cell surface), human PBMC cells, Fig 1B.
18.

Molecular cloning and characterization of equine Toll-like receptor 9. Zhang YW, EG Davis, F Blecha, MJ Wilkerson. Veterinary Immunology and Immunopathology 124: 208-219 (2008)..

Imgenex Products cited for Flow (Cell Surface) and Flow (Intracellular):.

IMG-305C (TLR9-FITC) and IMG-305D (TLR9-PE): Fig 2B (HEK293 and equine TLR9 transfected cells), Fig 4 (equine leukocytes), Fig 5 (equine PHA-stimulated PBMC), Fig 6 (equine leukocyte subsets). .

Notes: TLR9 expression was found to be predominantly intracellular. HEK293 cells express endogenous TLR9. TLR9 expression increased in equine TLR9 transiently transfected HEK293 cells. TLR9 expression increased in PBMC cells following PHA stimulation.  

19.
Antibodies specific for human or murine Toll-like receptors detect canine leukocytes by flow cytometry. Burgener IA and TW Jungi. Veterinary Immunology and Immunopathology124:184-191 (2008). Imgenex products cited for canine (dog) PBMC subpopulations:
1. 10083K [IC-Flow (Intracellular Staining Flow Assay) Kit
2. IMG-416A (TLR2), Flow (Cell Surface), Fig 1 and Tables 1 & 2
3. IMG-315A (TLR3), Flow (Intracellular), Tables 1 & 2
4. IMG-417A (TLR4), Flow (Intracellular), Fig 1 and Tables 1 & 2
5. IMG-663A (TLR5), Flow (Cell Surface), Fig 1 and Tables 1 & 2
6. IMG-664A (TLR5), Flow (Cell Surface), Tables 1 & 2
7. IMG-305A (TLR9), Flow (Intracellular), Fig 2 and Tables 1 & 2

20. Inhibitors of TLR8 reduce TNF production from human rheumatoid synovial membrane cultures. Sacre S, A Lo, B Gregory, R Simmonds, L Williams, M Feldmann, F Brennan, B Foxwell. J Immunol 181: 8002-8009 (2008). Imgenex antibodies cited [Flow (intracellular), human synovial membrane cells, Fig. 4B]:
1. TLR3-FITC (IMG-315C)
2. TLR8-FITC (IMG-321C)
3. TLR9-FITC (IMG-305C).

21. Role for plasmacytoid dendritic cells in the immune control of recurrent human herpes simplex virus infection. Donaghy H, L Bosnjak, A Harman, V Marsden, S Tyring, T Meng, A Cunningham.  Journal of Virology 83: 1952-1961 (2009). IMG-305C: Human dendritic cells,  Flow(cell surface), Fig. 3A(left); Flow (intracellular), Fig. 3A (right).
22.

Stimulation of iNOS expression and apoptosis resistance in B-cell chronic lymphocytic leukemia (B-CLL) cells through engagement of Toll-like receptor 7 (TLR-7) and NF-kB activation. Hammadi A, Billard C, A-M Faussat, J-P Kolb. Nitric Oxide 19:138-145 (2008).Imgenex products cited (B-CLL cells from a leukemia patient):

1.  IMG-417E (TLR4-FITC, clone HTA125): Flow (Intracellular), Fig 1: TLR4 was undetectable in the B-CLL cells.

2. IMG-305C (TLR9-FITC, clone 26C593.2): Flow (intracellular), Fig 1: TLR9 was detected in the BCLL cells

3. IMK-501 (Phospho-IkBa ActivELISA Kit): Fig 6B, ELISA data shows increased of IkBa phosphoryation following resiquimod treatment which stimulates NF-kB activation. 
 
Note: No expression of TLR-4 or TLR-9 was detected at the surface of  B-CLL cells by Flow (cell surface).  

23. Deoxynucleic acids from Cryptococcus neoformans activate myeloid dendritic cells via a TLR9-dependent pathway. Nakamura K, A Miyazato, G Xiao, M Hatta, K Inden, T Aoyagi, K Shiratori, K Takeda, S Akira, S Saijo, Y Iwakura, Y Adachi, N Ohno, K Suzuki, J Fujita, M Kakau, and K Kawakami.  J Immunol 180:4067-4074 (2008). Imgenex antibody used: TLR9-FITC (IMG-305C) for IF/ICC (intracellular confocal microscopy) in the following figures:
1. Fig 8a: primary mouse bone marrow-derived myeloid dendritic cells (BM-DC) treated with Cn-DNA and CpG-Rhodamine and analyzed over a time course
2. Fig 8b: BM-DC and RAW264.1 cells treated with Cn-DNA  
Note: intracellular redistribution of TLR9 was observed in treated cells

24. Members of the Toll-like Receptor Family of Innate Immunity Pattern-Recognition Receptors Are Abundant in the Male Rat Reproductive Tract. Palladino MA., TA Johnson, R Gupta, JL Chapman, and P Ojha. Biol Reproduction., in press, DOI: 10.1095/biolreprod.106.059410 (2007). IMGENEX antibodies cited: The following antibodies were used for WB in Fig. 2 using rat, testis, epididymis, and vas deferens: TLR 1 (IMG-5012), TLR2 (IMG-545), TLR5 (IMG-580), TLR6 (IMG-527), TLR7 (IMG-581) TLR9 (IMG-305A), TLR10 (IMG-5255A), TLR11 (IMG-5034) and MYD88 (IMG-178).
25. The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Koichi Tabeta, Kasper Hoebe, Edith M Janssen, Xin Du, Philippe Georgel, Karine Crozat, Suzanne Mudd, Navjiwan Mann, Sosathya Sovath, Jason Goode, Louis Shamel, Anat A, et al. Nature Immunology 7, 156-164 (2006). Imgenex antibodies cited: 1. TLR9 (IMG-305) [IF/ICC, Fig.5g-i (primary mouse macrophages)]. The specificity of the TLR9 antibody is demonstrated using primary mouse macrophages from wildtype (5g) and TLR9 knockout (5i) mice. 

Research purposes only. Not for diagnostic or in vivo use. This product is guaranteed to perform as indicated on the datasheet for six months from the date of purchase.

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