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Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (PE Conjugate) #14575
Gallery: Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (PE Conjugate) #14575
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 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.
- 50% methanol.
- 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
- Aliquot 100 μl fresh whole blood per assay tube.
- OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
- Add 65 μl of 10% formaldehyde to each tube.
- Vortex briefly and let stand for 15 min at room temperature.
- Add 1 ml of 0.1% Triton™ X-100 to each tube.
- Vortex and let stand for 30 min at room temperature.
- Add 1 ml incubation buffer.
- Pellet cells by centrifugation and aspirate supernatant.
- Repeat steps 7 and 8.
- Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
- Incubate at least 10 min on ice.
- 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.
- Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
- Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
- Incubate for 30–60 min at room temperature.
- Wash by centrifugation in 2–3 ml incubation buffer.
- 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
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
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro450 of human toll-like receptor 9 protein.
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).
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.