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14575
Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (PE Conjugate) 
Antibody Conjugates
Monoclonal Antibody

Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (PE Conjugate)  #14575

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Flow Cytometry Image 1 - Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (PE Conjugate) 

Flow cytometric analysis of human whole blood using Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (PE Conjugate) co-stained with either CD11c or CD3. CD11c+ dendritic cells are distinctly positive for toll-like receptor 9 while CD3+ T cells are negative.

To Purchase # 14575S
Product # Size Price
14575S
100 µl  (50 tests) $ 358

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb #13674.

Product Usage Information

Application Dilution
Flow Cytometry 1:50

Storage

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibodies. Protect from light. Do not freeze.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised June 2020

Protocol Id: 407

Specificity / Sensitivity

Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total toll-like receptor 9 protein. This antibody is predicted to recognize known full-length isoforms of toll-like receptor 9, but not cleaved toll-like receptor 9 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro450 of human toll-like receptor 9 protein.

Background

Members of the Toll-like receptor (TLR) family, named for the closely related Toll receptor in Drosophila, play a pivotal role in innate immune responses (1-4). TLRs recognize conserved motifs found in various pathogens and mediate defense responses (5-7). Triggering of the TLR pathway leads to the activation of NF-κB and subsequent regulation of immune and inflammatory genes (4). The TLRs and members of the IL-1 receptor family share a conserved stretch of approximately 200 amino acids known as the Toll/Interleukin-1 receptor (TIR) domain (1). Upon activation, TLRs associate with a number of cytoplasmic adaptor proteins containing TIR domains, including myeloid differentiation factor 88 (MyD88), MyD88-adaptor-like/TIR-associated protein (MAL/TIRAP), Toll-receptor-associated activator of interferon (TRIF), and Toll-receptor-associated molecule (TRAM) (8-10). This association leads to the recruitment and activation of IRAK1 and IRAK4, which form a complex with TRAF6 to activate TAK1 and IKK (8,11-14). Activation of IKK leads to the degradation of IκB, which normally maintains NF-κB in an inactive state by sequestering it in the cytoplasm.

Toll-like receptor 9 (TLR9) is highly expressed in macrophages, dendritic cells, and B lymphocytes; five isoforms are generated by alternative splicing in humans (15,16). TLR9 binds to unmethylated CpG motifs present on bacterial DNA and stimulates NF-κB via the MyD88 adaptor protein (17-19). In contrast to most TLR family members that are localized to the plasma membrane, TLR9 is an intracellular receptor localized to the ER in resting cells (20). Upon binding to CpG DNA, TLR9 is proteolytically processed and translocates to endo-lysosomal compartments where it binds MyD88 to initiate downstream signaling (21-23).

  1. Akira, S. (2003) J Biol Chem 278, 38105-8.
  2. Beutler, B. (2004) Nature 430, 257-63.
  3. Dunne, A. and O'Neill, L.A. (2003) Sci STKE 2003, re3.
  4. Medzhitov, R. et al. (1997) Nature 388, 394-7.
  5. Schwandner, R. et al. (1999) J Biol Chem 274, 17406-9.
  6. Takeuchi, O. et al. (1999) Immunity 11, 443-51.
  7. Alexopoulou, L. et al. (2001) Nature 413, 732-8.
  8. Zhang, F.X. et al. (1999) J Biol Chem 274, 7611-4.
  9. Horng, T. et al. (2001) Nat Immunol 2, 835-41.
  10. Oshiumi, H. et al. (2003) Nat Immunol 4, 161-7.
  11. Muzio, M. et al. (1997) Science 278, 1612-5.
  12. Wesche, H. et al. (1997) Immunity 7, 837-47.
  13. Suzuki, N. et al. (2002) Nature 416, 750-6.
  14. Irie, T. et al. (2000) FEBS Lett 467, 160-4.
  15. Du, X. et al. (2000) Eur Cytokine Netw 11, 362-71.
  16. Chuang, T.H. and Ulevitch, R.J. (2000) Eur Cytokine Netw 11, 372-8.
  17. Hemmi, H. et al. (2000) Nature 408, 740-5.
  18. Bauer, S. et al. (2001) Proc Natl Acad Sci U S A 98, 9237-42.
  19. Takeshita, F. et al. (2001) J Immunol 167, 3555-8.
  20. Latz, E. et al. (2004) Nat Immunol 5, 190-8.
  21. Park, B. et al. (2008) Nat Immunol 9, 1407-14.
  22. Ewald, S.E. et al. (2008) Nature 456, 658-62.
  23. Sepulveda, F.E. et al. (2009) Immunity 31, 737-48.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.
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XP is a registered trademark of Cell Signaling Technology, Inc.