title: Comprehensive Analysis of Immunological Synapse Phenotypes Using Supported Lipid Bilayers.
abstract: Supported lipid bilayers (SLB) formed on glass substrates have been a useful tool for study of immune cell signaling since the early 1980s. The mobility of lipid-anchored proteins in the system, first described for antibodies binding to synthetic phospholipid head groups, allows for the measurement of two-dimensional binding reactions and signaling processes in a single imaging plane over time or for fixed samples. The fragility of SLB and the challenges of building and validating individual substrates limit most experimenters to ~10 samples per day, perhaps increasing this few-fold when examining fixed samples. Successful experiments might then require further days to fully analyze. We present methods for automation of many steps in SLB formation, imaging in 96-well glass bottom plates, and analysis that enables >100-fold increase in throughput for fixed samples and wide-field fluorescence. This increased throughput will allow better coverage of relevant parameters and more comprehensive analysis of aspects of the immunological synapse that are well reconstituted by SLB.
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford, Oxford, OX3 7FY, UK.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Valvo
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford, Oxford, OX3 7FY, UK.
authors.forename: Viveka
authors.initials: V
authors.lastname: Mayya
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, The University of Oxford, Oxford, OX3 7FZ, UK.
authors.forename: Elena
authors.initials: E
authors.lastname: Seraia
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford, Oxford, OX3 7FY, UK.
authors.forename: Jehan
authors.initials: J
authors.lastname: Afrose
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford, Oxford, OX3 7FY, UK.
authors.forename: Hila
authors.initials: H
authors.lastname: Novak-Kotzer
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, The University of Oxford, Oxford, OX3 7FZ, UK. daniel.ebner@ndm.ox.ac.uk.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ebner
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford, Oxford, OX3 7FY, UK. michael.dustin@kennedy.ox.ac.uk.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Dustin
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D054992:Immunological Synapses / Q000737:chemistry* / Q000276:immunology; D008051:Lipid Bilayers / Q000737:chemistry* / Q000276:immunology
chemical_list: D008051:Lipid Bilayers
keywords: Costimulation; High-throughput screening; Image analysis; Immunological synapse; Signaling; Supported lipid bilayers