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14575S 100 µl (50 tests) $329.00


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H Endogenous Rabbit IgG

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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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. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Conjugated Primary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 30–60 min at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

Protocol Id: 384

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.

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