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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Flow Cytometry

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.

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

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

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 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. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  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 antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
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.

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.

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.


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.  Chuang, T.H. and Ulevitch, R.J. (2000) Eur Cytokine Netw 11, 372-8.

16.  Du, X. et al. (2000) Eur Cytokine Netw 11, 362-71.

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.


Entrez-Gene Id 54106
Swiss-Prot Acc. Q9NR96


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

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