Supported Lipid Bilayer Technology for the Study of Cellular Interfaces.
Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell-antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology.
Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.|These authors contributed equally to this work.
Travis J
TJ
Crites
Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.|These authors contributed equally to this work.
Michael
M
Maddox
Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Kartika
K
Padhan
Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.|Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, Department of Pathology, New York University School of Medicine, New York, New York.
James
J
Muller
Flow Cytometry Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Calvin
C
Eigsti
Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Rajat
R
Varma
D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D008051:Lipid Bilayers; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D011994:Recombinant Proteins
CHOs cells; diffusion; liposome extrusion; protein engineering; recombinant transmembrane proteins; signal transduction; supported lipid bilayers; suspension CHO cells; synapse