Measuring TLR function in transfectants.
This unit summarizes a combination of methods that can be optimized for measuring toll-like receptor (TLR) function. TLRs serve as primary innate immune sensors and exhibit high specificity towards evolutionarily conserved microbial and viral structures. The unit focuses specifically on TLR4, the principal Gram-negative lipopolysaccharide (LPS) sensor. Methods described include transient transfections, analyses of activation of various promoters in reporter-gene assays, and induction of IL-8 secretion. Other topics that will be briefly discussed include the necessity for the assessment of surface expression of transmembrane receptors (e.g., TLR4) using FACS analysis, and a permutation of the TLR functional analysis approach using site-directed mutagenesis.
University of Maryland, Baltimore, Maryland, USA.
Prasad
P
Rallabhandi
D000818:Animals; D002469:Cell Separation; D005434:Flow Cytometry; D006090:Gram-Negative Bacteria / Q000276:immunology*; D006801:Humans; D007113:Immunity, Innate; D007158:Immunologic Techniques; D008070:Lipopolysaccharides / Q000276:immunology; D008967:Molecular Biology / Q000379:methods; D016297:Mutagenesis, Site-Directed; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051193:Toll-Like Receptors / Q000276:immunology*
D008070:Lipopolysaccharides; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors