Intracellular Staining by Flow Cytometry
|Detection of TLR9 in Human CD123+ PBMCs by Flow Cytometry. Human CD123+ peripheral blood mononuclear cells (PBMCs) were stained with Sheep Anti-Human TLR9 Alexa Fluor® 488‑conjugated Antigen Affinity-purified Polyclonal Antibody (Catalog # IC7108G, filled histogram) or isotype control antibody (Catalog # IC016G, open histogram). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
TLR9 (Toll receptor 9; also CD289) is a 145-150 kDa member of the Toll-like receptor family of molecules. It is expressed by colonic epithelium, CD123+ plasmacytoid dendritic cells, and transitional B cells, and responds to unmethylated DNA CpG motifs that exhibit either a GTCGTT sequence (in human), or a GACGTT sequence (in mouse). TLR9 is found in the ER, and translocates to either the cell membrane, or to lysosomes where it binds bacterial DNA. Precursor human TLR9 is a type I transmembrane protein 1032 amino acids (aa) in length. It possesses a 793 aa extracellular region that contains 26 LRRs (aa 26-818), plus a 193 aa cytoplasmic domain. The full-length 150 kDa form is suggested to be ligand-binding but nonsignaling. The active form is believed to be an 80 kDa cleavage product found in the endosome compartment. There are multiple splice forms. One contains a deletion of aa 2-16, a second possesses an alternate start site at Met58, while a third and fourth show alternative start sites aa 23 and 24 upstream of the standard site. Over aa 64-189, human TLR9 shares 76% aa identity with mouse TLR9.