Improved method of preparation of supported planar lipid bilayers as artificial membranes for antigen presentation

Abstract

T cell activation is the result of direct cell-cell contact between T cells and antigen presenting cells (APCs), and of interactions between membrane-bound ligands and receptors at the contact interface, the "immunological synapse." Model APCs based upon supported fluid lipid bilayers have been used to dissect these complex molecular interactions and to facilitate real-time microscopic observations. Nearly all studies have used liposome fusion-based methods to make supported bilayers, and the biophysical properties of these membranes were not characterized in detail. Here, using both Langmuir-Blodgett and liposome fusion techniques, we explored five different methods of lipid bilayer preparation on glass, mica, or dextran cushion substrates and characterized the stability, homogeneity, and fluidity of the bilayers with fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Most combinations of techniques and substrates led to unsatisfactory results, notably, a lack of homogeneity for liposome fusion on glass, low stability of bilayers on mica, and loss of fluidity of dextran-cushioned bilayers in solutions containing protein. To overcome these deficits, we developed a technique that combines liposome fusion on glass and thermally enhanced bilayer expansion. The newly expanded pristine bilayer showed high degrees of stability, homogeneity, and fluidity. MHC and ICAM-1 molecules anchored on the bilayer diffused freely and stimulated T cell calcium flux and adhesion, respectively.

Ligand-Mediated Friction Determines Morphodynamics of Spreading T Cells

“…S5 B). This last observation is consistent with earlier reports (35). Single-particle tracking provides on Fix an upper bound for D x 0.001 mm 2 /s (Fig.…”

Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in Supported Lipid Bilayer Using a Single Nanopore Chip

“…Supported lipid membranes (SLMs) ,− have been introduced and reported for developing biosensors with long-term stability. , The most commonly used techniques to produce SLMs on hydrophilic substrates include Langmuir–Blodgett followed by Langmuir–Schaefer (LB/LS), − the vesicle adsorption and rupture, ,− combined LB and liposome fusion, and droplet-interface bilayer. − SLMs based on these techniques have shown more stability as compared with BLMs. , However, the usability of these membranes is constrained by their close surface proximity, which substantially averts the fusion of integral proteins. Lipid bilayer produced on a solid support may not be consider as a significant apparatus to explore the electrophysiology of transmembrane proteins because of excessive ions present near the substrate and may denature the protein channels embedded in SLM .…”

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy

“…Supported lipid bilayers (SLBs) are commonly used to model cell membranes and its native proteins [ 1 ]. Production of SLBs as a model system comprised of either single or multiple component lipids has been done using vesicle fusion [ 2 , 3 , 4 , 5 , 6 , 7 ], Langmuir–Blodgett/Langmuir–Schaefer deposition [ 8 , 9 ]. Scanning probe lithography methods as Dip-Pen Nanolithography [ 10 , 11 , 12 , 13 ] and Polymer Pen Lithography [ 14 ] could also be utilized for the production of lipid bilayer membrane.…”