Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

Travis J. Crites1, Michael Maddox1, Kartika Padhan2, James Muller3, Calvin Eigsti4, Rajat Varma2

1 These authors contributed equally to this work, 2 Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 3 Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, Department of Pathology, New York University School of Medicine, New York, New York, 4 Flow Cytometry Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 24.5
DOI:  10.1002/0471143030.cb2405s68
Online Posting Date:  September, 2015
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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.

Keywords: supported lipid bilayers; synapse; signal transduction; diffusion; protein engineering; liposome extrusion; recombinant transmembrane proteins; suspension CHO cells; CHOs cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Procedure for Making Liposomes of Ni‐NTA or CAP‐Biotin Lipids Using Extrusion
  • Basic Protocol 2: Procedure for Making Lipid Bilayers in Flow Cells
  • Basic Protocol 3: Procedure for Studying the Immune Synapse Formed Between Jurkat Cells and Supported Lipid Bilayers Containing α‐CD3 Antibodies and ICAM‐1
  • Basic Protocol 4: Procedure for Preparing Lipid Bilayers on Silica Beads
  • Basic Protocol 5: Stimulating T Cells with Artificial APCs
  • Basic Protocol 6: Design, Construction, and Purification of Tailless Transmembrane‐Anchored Proteins for Incorporation in Lipid Bilayers
  • Support Protocol 1: Culturing and Transfection of CHOs Cells for the Expression of Poly‐His Tagged, GPI‐Anchored, and Transmembrane‐Anchored Proteins
  • Support Protocol 2: Phosphatidylinositol‐Specific Phospholipase C Treatment for Test of GPI Linkage
  • Support Protocol 3: Preparation of Casein Reagent
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Procedure for Making Liposomes of Ni‐NTA or CAP‐Biotin Lipids Using Extrusion

  Materials
  • Tris‐saline buffer (see recipe)
  • Nitrogen gas cylinder with regulator
  • High‐purity chloroform
  • 10 mg/ml Ni‐NTA (1,2‐dioleoyl‐sn‐glycero‐3‐[(N‐(5‐amino‐1‐carboxypentyl)iminodiacetic acid)succinyl]) [nickel salt] lipid stocks (Avanti Polar Lipids, Inc.) in chloroform
  • 10 mg/ml DOPC (1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine) lipid stocks (Avanti Polar Lipids) in chloroform
  • 10 mg/ml CAP‐biotin (1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(CAP biotinyl)) lipid stocks (Avanti Polar Lipids) in chloroform
  • Argon gas cylinder with regulator
  • Gas dispersion tube with fritted end (Sigma, cat. no. CLS3953312C)
  • Screw‐top glass vials
  • Lyophilizer
  • Mini‐extruder kit, including retainer nut, extruder outer casing, Teflon bearing, internal membrane supports, O‐rings, filter supports, air‐tight 1‐μl glass syringes, extruder stand, and extrusion membranes (0.1 μm in size) (Avanti Polar Lipids)
  • Autoclaved glass beaker
  • Bath sonicator

Basic Protocol 2: Procedure for Making Lipid Bilayers in Flow Cells

  Materials
  • HBS‐BSA (see recipe)
  • 1× Ni‐NTA or CAP‐biotin liposome solutions ( protocol 1)
  • Poly‐His‐tagged or streptavidin‐modified proteins of interest (the protein of interest would typically be a ligand of a receptor expressed on the cell type of interest)
  • Casein solution (see protocol 9)
  • 20‐ml plastic syringes
  • Focht Chamber System (FCS2) (Bioptechs; http://www.bioptechs.com/Products/FCS2/fcs2.html)
  • Piranha solution–etched 40‐mm‐diamter cover glasses (Dustin et al., )
  • Tygon tubing (R‐3603; i.d. 1/16 in., o.d. 1/8 in.)
  • Two‐way and three‐way stopcocks (BioRad)

Basic Protocol 3: Procedure for Studying the Immune Synapse Formed Between Jurkat Cells and Supported Lipid Bilayers Containing α‐CD3 Antibodies and ICAM‐1

  Materials
  • 1× Ni‐NTA and 1× CAP‐biotin liposome solutions ( protocol 1)
  • Mono‐biotinylated α‐CD3 (Fleire and Batista, )
  • Streptavidin
  • Poly‐His‐tagged ICAM‐1 (Sino Biological, cat. no. 50440‐M08H‐20)
  • Casein solution (See protocol 9)
  • HBS‐BSA (see recipe)
  • Additional reagents and equipment for preparing glass‐supported lipid bilayers in flow cells ( protocol 2)

Basic Protocol 4: Procedure for Preparing Lipid Bilayers on Silica Beads

  Materials
  • 1× Ni‐NTA and 1× CAP‐biotin liposome solutions ( protocol 1)
  • 5‐μm silica bead suspension (supplied as 10% solid; Bangs Laboratories, cat. no. SS05N)
  • HBS‐BSA (see recipe)
  • Proteins of interest
  • 96‐well V‐bottom microtiter plate or 1.5‐ml microcentrifuge tubes
  • Plate‐sealing tape
  • Benchtop centrifuge with microtiter plate adapter
  • Orbital microtiter plate shaker

Basic Protocol 5: Stimulating T Cells with Artificial APCs

  Materials
  • T cells, or other cells used for stimulation (see appropriate units of Coligan et al., )
  • Artificial APCs ( protocol 4)
  • FACS buffer (see recipe), chilled
  • Primary antibodies against the extracellular portion of the proteins of interest or against intracellular proteins or phosphorylated proteins of interest
  • Fluorescently labeled secondary antibodies against primary antibody isotype, if necessary
  • FACS buffer (see recipe) containing 0.05% (w/v) saponin
  • 2% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Lysis buffer (see recipe)
  • 6× SDS loading dye (unit 6.1; Gallagher, )
  • Molecular weight markers for SDS‐PAGE (also see unit 6.1; Gallagher, )
  • 5% (w/v) nonfat dry milk in PBS (see appendix 2A for PBS)
  • Flat‐bottom microtiter plate, number of wells determined by Table 24.5.1
  • Benchtop centrifuge with microtiter plate adapters
  • 37°C water bath or 37°C, 5% CO 2 humidified incubator
  • 2‐ml U‐bottom centrifuge tubes
  • Boiling water bath
  • Nitrocellulose membranes (see unit 6.1; Gallagher, )
  • Additional reagents and equipment for flow cytometry (Robinson et al., ), SDS‐PAGE (unit 6.1; Gallagher, ), and western blotting (immunoblotting; unit 6.2; Gallagher et al., )

Basic Protocol 6: Design, Construction, and Purification of Tailless Transmembrane‐Anchored Proteins for Incorporation in Lipid Bilayers

  Materials
  • CD CHO medium (Life Technologies)
  • HT supplement (Life Technologies)
  • 100× penicillin‐streptomycin (e.g., Life Technologies)
  • L‐glutamine (e.g., Life Technologies)
  • Phenol red (e.g., Life Technologies; optional)
  • Suspension CHO (CHOs) cells (Life Technologies)
  • Amaxa transfection Kit V, containing transfection solution V, transfection cuvettes, and plastic droppers (Lonza)
  • Plasmid DNA construct for protein to be expressed, including G418 (or similar) resistance cassette
  • G418 sulfate (e.g., Life Technologies)
  • Clumping reagent (Life Technologies; optional)
  • Hanks’ balanced salt solution (HBSS; see recipe in appendix 2A or use commercial version) containing 5% bovine serum albumin (BSA) and 1× penicillin‐streptomycin
  • 6‐well flat bottom cell culture plate
  • 37°C humidified 5% CO 2 incubator with orbital shaker
  • Conical centrifuge tubes (e.g., Corning Falcon)
  • 125‐ml (Corning, cat. no. 431143) and 250‐ml (Corning, cat. no. 431144) conical culture flasks
  • Amaxa Nucleofector™ 2b device (Lonza)
  • 50‐μm nylon mesh filter
  • FACS tubes
  • Fluorescence‐activated cell sorter (FACS)
  • Additional reagents and equipment for basic cell culture techniques including determining cell viability by trypan blue exclusion (unit 1.1; Phelan and May, ), and fluorescence‐activated cell sorting (Robinson et al., )

Support Protocol 1: Culturing and Transfection of CHOs Cells for the Expression of Poly‐His Tagged, GPI‐Anchored, and Transmembrane‐Anchored Proteins

  Materials
  • CHOs cells or other mammalian cell line
  • Amaxa transfection Kit V, containing transfection solution V, transfection cuvettes, and plastic droppers (Lonza)
  • Phosphatidylinositol‐specific phospholipase C (PIPLC; from Bacillus cereus; Sigma, cat. no. P5542)
  • 10 mM Tris·Cl, pH 7.4 ( appendix 2A), containing 144 mM NaCl and 0.05% (w/v) BSA
  • Plasmid DNA encoding CD80‐TM and CD80‐CD28‐TM [cDNA encoding CD80‐TM and CD80‐CD28‐TM were generated according to the steps outlined in protocol 6; these constructs are available as a courtesy from our lab; please contact Rajat Varma ( )]
  • HBS‐BSA (see recipe)
  • FACS buffer (see recipe), cold
  • Amaxa Nucleofector™ 2b device (Lonza)
  • 24‐well plates
  • Refrigerated centrifuge with microtiter plate adapter
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Support Protocol 2: Phosphatidylinositol‐Specific Phospholipase C Treatment for Test of GPI Linkage

  Materials
  • Casein
  • 10 N NaOH
  • 10× phosphate‐buffered saline (PBS; see recipe for 1× PBS in appendix 2A)
  • 500‐ml plastic bottle with screw cap
  • Magnetic stir plate and stir bar
  • pH meter
  • Ultracentrifuge with Beckman 45 Ti rotor and appropriate ultracentrifuge tubes
  • Ring stand with clamps
  • 250‐ml 0.22‐μm Millipore Stericup filter
  • FACS tubes
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Figures

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Literature Cited

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Key References
  Dustin et al., 2007. See above.
  These are key supporting references for this unit. The first reference provides a detailed step‐by‐step procedure for purifying GPI‐anchored proteins and their subsequent incorporation in liposomes. We have followed the same protocol for the purification of tailless transmembrane‐anchored proteins described in Basic Protocol 6 and their incorporation in liposomes following purification. The second reference provides a video protocol for formation of bilayers in FCS2 perfusion chambers described here in . Both references contain important steps on how to clean cover glasses with Piranha solution for formation of lipid bilayers.
  Vardana and Dustin, 2008. See above
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