Making Giant Unilamellar Vesicles via Hydration of a Lipid Film

Suliana Manley1, Vernita D. Gordon2

1 National Institutes of Health, Cell Biology and Metabolism Branch, Bethesda, Maryland, 2 University of Illinois Urbana‐Champaign, Department of Materials Science and Engineering, Urbana, Illinois
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 24.3
DOI:  10.1002/0471143030.cb2403s40
Online Posting Date:  September, 2008
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Abstract

This unit describes protocols for making giant unilamellar vesicles (GUVs) based on rehydration of dried lipid films. These model membranes are useful for determining the impact of membrane and membrane‐binding components on lipid bilayer stiffness and phase behavior. Due to their large size, they are especially amenable to studies using fluorescence and light microscopy, and may also be manipulated for mechanical measurements with optical traps or micropipets. In addition to their use in encapsulation, GUVs have proven to be useful model systems for studying many cellular processes, including tubulation, budding, and fusion, as well as peptide insertion. The introduction of enzymes or proteins can result in reorganization, leading to such diverse behavior as vesicle aggregation, fusion, and fission. Curr. Protoc. Cell Biol. 40:24.3.1‐24.3.13. © 2008 by John Wiley & Sons, Inc.

Keywords: liposomes; giant vesicles; electroformation; liposome swelling; model membranes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing GUVs by Electroformation on Indium Tin Oxide–Coated Plates
  • Support Protocol 1: Making Lipid Mixtures
  • Support Protocol 2: Design of a PTFE (Teflon) Holder for Electroformation on ITO‐Coated Plates
  • Alternate Protocol 1: Preparing GUVs by Electroformation on Platinum Wires
  • Support Protocol 3: Design of a Chamber for Electroformation on Platinum Wires
  • Basic Protocol 2: Preparing GUVs by Swelling off of PTFE (Teflon)
  • Alternate Protocol 2: Preparing GUVs by Swelling Off a Uniform Film on Glass
  • Alternate Protocol 3: Preparing GUVs by Dehydration and Rehydration of SUVs
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing GUVs by Electroformation on Indium Tin Oxide–Coated Plates

  Materials
  • 100 µl lipid in chloroform, at 5 to 10 mg/ml (see protocol 2)
  • Up to 600 mM sucrose, or sterile deionized H 2O
  • 25‐cm2 ITO‐coated plates (e.g., Delta Technologies, Ltd., part no. CG‐511IN‐50x50/1.1; http://www.delta‐technologies.com/)
  • Sealed chamber (e.g., vacuum desiccator) connected to dry nitrogen or vacuum source
  • Vacuum grease
  • Teflon holder and spacers for ITO‐coated plates (see protocol 3)
  • Oven with hole to introduce electrodes or heating bath (for lipid systems in which at least one component has a chain‐melting transition temperature, T m, above room temperature)
  • Electrodes (“Mini‐Plunger to BNC Male” or equivalent; e.g., Radio Shack)
  • Function generator with readout for voltage and frequency (Stanford Research Systems, Ltd., http://www.thinksrs.com/)

Support Protocol 1: Making Lipid Mixtures

  Materials
  • Lipid (Avanti Polar Lipids; when received, store at –20°C or lower)
  • Chloroform
  • Methanol (optional)
  • Argon or nitrogen gas
  • Glass vials with Teflon closures
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • Teflon tape

Support Protocol 2: Design of a PTFE (Teflon) Holder for Electroformation on ITO‐Coated Plates

  • 5 to 10 µl of lipid solution in chloroform ( protocol 2) at concentration of 0.5 to 0.66 mM (∼0.5 mg/ml)
  • Up to 600 mM sucrose, or sterile deionized H 2O
  • Platinum wires, 0.5‐ to 2.0‐mm diameter
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • Sealed chamber (e.g., vacuum desiccator) connected to dry nitrogen or vacuum source
  • Electroformation chamber (see protocol 5), assembled with platinum wires

Alternate Protocol 1: Preparing GUVs by Electroformation on Platinum Wires

  Materials
  • Platinum wires, 0.5‐ to 2.0‐mm diameter
  • Aluminum block (machined; typical dimensions, 10 × 4 ×1 cm)
  • Electrical block fittings or terminal block fittings (McMaster‐Carr, http://www.mcmaster.com)
  • Optional items:
    • PTFE seals
    • Cover glass for microscopy
    • Norland optical adhesive
    • Resistive heating wire
    • Ceramic insulators and fittings for electrical attachment
    • Thermocouple

Support Protocol 3: Design of a Chamber for Electroformation on Platinum Wires

  Materials
  • 20 mg/ml lipid in chloroform ( protocol 2)
  • Chloroform
  • Nitrogen gas
  • Swelling solution: 100 mM sucrose or glucose prepared using sterile deionized H 2O
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • PTFE (Teflon) sheet, ∼2 × 2–cm square, roughened with fine‐grain sandpaper (McMaster‐Carr, http://www.mcmaster.com)
  • Desiccator chamber, attached to vacuum pump
  • Glass beaker
  • Incubator or oven

Basic Protocol 2: Preparing GUVs by Swelling off of PTFE (Teflon)

  Materials
  • 80 µl lipids in chloroform ( protocol 2) at 10 mM, (∼10 mg/ml)
  • Chloroform
  • Nitrogen gas
  • Swelling solution: 100 mM sucrose in water (or other aqueous swelling solution), prepared using sterile deionized H 2O
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • 50‐ to 100‐ml glass flask with pointed bottom
  • Rotary evaporator
  • Vacuum oven
  • Oven or incubator

Alternate Protocol 2: Preparing GUVs by Swelling Off a Uniform Film on Glass

  Materials
  • 100 to 300 µl of lipid solution in chloroform at 20 mg/ml ( protocol 2)
  • 100 mM NaCl (prepared using sterile deionized H 2O) containing 1 vol% glycerol
  • 100 mM NaCl (prepared using sterile deionized H 2O), without glycerol
  • Hamilton syringe
  • Small glass vial
  • Desiccator chamber, attached to vacuum pump
  • Bath sonicator (43 kHz; L&R Ultrasonics Model T21, http://www.lrultrasonics.com)
  • Glass coverslips
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Figures

Videos

Literature Cited

Literature Cited
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   Bacia, K., Schwille, P., and Kurzchalia, T.V. 2005. Sterol structure determines the separation of phases and the curvature of the liquid‐ordered phase in model membranes. Proc. Natl. Acad. Sci. U.S.A. 102:3272‐3277.
   Bagatolli, L.A. 2006. To see or not to see: Lateral organization of biological membranes and fluorescence microscopy. Biochim. Biophys. Acta 1758:1541‐1556.
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