Intracellular Staining by Flow Cytometry
Detection of TLR3 in A549 Human Cell Line by Flow Cytometry.A549 human lung carcinoma cell line was stained with Goat Anti-Human TLR3 APC‑conjugated Antigen Affinity-purified Polyclonal Antibody (Catalog # IC1487A, filled histogram) or isotype control antibody (Catalog # IC108A, 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.
Human 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 human TLR3 cDNA encodes a 904 amino acid (aa) precursor that contains a 23 aa signal sequence, a 681 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 human TLR3 shares 80%, 79%, and 77% aa sequence identity with the ECD of rat, mouse, 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). TLR3 is expressed in dendritic cells, macrophages, microglia, and astrocytes (13-15) and is upregulated by IFN-beta and LPS (9, 14). TLR3 expression is also induced by lung fibroblasts and epithelial cells by respiratory syncytial virus infection (12).