Intracellular Staining by Flow Cytometry
|Detection of TLR3 in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes were stained with Rat Anti-Mouse TLR3 PE‑conjugated Monoclonal Antibody (Catalog # IC3005P, filled histogram) or isotype control antibody (Catalog # IC006P, 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.|
Mouse TLR3 is a 116 kDa type I transmembrane glycoprotein that belongs to the mammalian Toll-Like Receptor family of pathogen pattern recognition molecules (1, 2). There are at least eleven mouse and ten human members that activate the innate immune system following exposure to a variety of microbial species (3). The mouse TLR3 cDNA encodes a 905 amino acid (aa) precursor that contains a 25 aa signal sequence, a 680 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 179 aa cytoplasmic region (4). The horseshoe shaped ECD (5, 6) contains 23 leucine-rich repeats, and the cytoplasmic domain contains one Toll/IL-1 receptor (TIR) domain. The ECD of mouse TLR3 shares 90%, 79%, and 77% aa sequence identity with the ECD of rat, human, and bovine TLR3, respectively. TLR3 is found in phagosomes (7), where the acidic pH enables binding of internalized double stranded RNA and mRNA from viruses, parasites, and necrotic virally-infected cells (8‑11). Ligand binding by TLR3 induces receptor dimerization (5, 6, 8), leading to the release of inflammatory cytokines and dendritic cell maturation (9, 11‑13). Mouse TLR3 is expressed in dendritic cells, macrophages, microglia, and astrocytes (13‑15), and is up‑regulated by IFN-beta and LPS (9, 14). TLR3 expression is also induced on lung fibroblasts and epithelial cells by respiratory syncytial virus infection (12).
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