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.
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.
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.
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.
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.
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.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
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.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro450 of human toll-like receptor 9 protein.
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).
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