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Comprehensive Analysis of Immunological Synapse Phenotypes Using Supported Lipid Bilayers

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1584)

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.

Key words

Immunological synapse Supported lipid bilayers High-throughput screening Image analysis Costimulation Signaling 
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Notes

Acknowledgments

The authors thank M. Santos and S. Davis for sharing methods for protein production using the lentiviral system. Wellcome Trust grant 100262/Z/12/Z and the Kennedy Trust for Rheumatology Research supported this work.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal SciencesThe University of OxfordOxfordUK
  2. 2.Target Discovery Institute, Nuffield Department of MedicineThe University of OxfordOxfordUK

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