title: A high-content analysis toolbox permits dissection of diverse signaling pathways for T lymphocyte polarization.
abstract: RNA interfering (RNAi) screening strategies offer the potential to elucidate the signaling pathways that regulate integrin and adhesion receptor-mediated changes in T lymphocyte morphology. Of crucial importance, however, is the definition of key sets of parameters that will provide accurate, quantitative, and nonredundant information to flag relevant hits in such assays. In this study, the authors have used an image-based high-content analysis (HCA) technology platform and a panel of 24 pharmacological inhibitors, at a range of concentrations, to define key sets of parameters that enables sensitive and quantitative effects on integrin (LFA-1)-mediated lymphocyte morphology to be evaluated. In particular, multiparametric analysis of lymphocyte morphology that was based on intracellular staining of both the F-actin and alpha-tubulin cytoskeleton resulted in improved ability to discriminate morphological behavior compared to F-actin staining alone. Morphological and fluorescence intensity/distribution profiling of pharmacologically treated lymphocytes stimulated with integrin (LFA-1) and adhesion receptors (CD44) also revealed notable differences in their sensitivity to inhibitors. The assay described here may be used in HCA strategies such as RNAi screening assays to elucidate the signaling pathways and molecules that regulate integrin/adhesion receptor-mediated T lymphocyte polarization.
authors.affiliation: Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College, Dublin, Ireland.
authors.forename: Michael
authors.initials: M
authors.lastname: Freeley
authors.forename: Gabor
authors.initials: G
authors.lastname: Bakos
authors.forename: Anthony
authors.initials: A
authors.lastname: Davies
authors.forename: Dermot
authors.initials: D
authors.lastname: Kelleher
authors.forename: Aideen
authors.initials: A
authors.lastname: Long
authors.forename: Dara J
authors.initials: DJ
authors.lastname: Dunican
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
chemical_list: D000199:Actins; D016169:Lymphocyte Function-Associated Antigen-1; D014404:Tubulin