Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

Abstract

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. © 2015 by John Wiley & Sons, Inc.

Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles

“…Eight chamber glass slides (1.5 Lab-Tek II; Nunc) were coated with 0.01% poly- l -lysine (PLL) and dried at 60 °C for 1 h. Slides were coated with His-ICAM-1 (2.5 µg/mL; produced in house) alone or with His-MICA (2.5 µg/mL; Sino Biological), B7-H6-Fc (2.5 µg/mL; R&D Systems), αNKG2A (5 µg/mL; R&D Systems), or αNKp30 (10 µg/mL; P30-15; Biolegend or 210845; R&D Systems) in phosphate-buffered saline (PBS) overnight (4 °C). Bilayers were prepared as previously described ( 8 ) and functionalized with His-ICAM-1 (2.5 µg/mL) alone or with His-MICA (2.5 µg/mL), biotinylated-αNKp30 (10 µg/mL; P30-15), or biotinylated-αCD3 (5 µg/mL; OKT3; a gift from Andy Shepherd, GSK).…”

Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells

“…SUV are defined as vesicles in the 20–100 nm range. SUV were formed by extrusion as described using the Avanti Miniextruder with a 100 nm filter (Crites et al, 2015). When SUV were used to mimic SE, all lipids were combined prior to SUV formation, whereas BSLB and PSLB composition could be determined by mixing different proportions of stock SUVs as the final bilayer composition is determined by the average of the input SUV.…”

Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner

“…Three wash steps were performed between each addition. Activation of OT1 T cells on glass-supported planar lipid-bilayer was performed as described previously [ 45 , 50 ]. In brief, biotin-CAP-PE and DGS-Ni were passed through a mini-extruder kit (Avanti Polar Lipids, Alabaster, AL) to generate liposomes, which were then mixed and added to a flow chamber formed between a clean glass slide and a cover slip.…”