Membrane flow patterns in multicomponent giant vesicles induced by alternating electric fields

GUVs were prepared using the electroformation method, introduced by Angelova and Dimitrov. Stock solutions of 4 mM lipid in chlorophorm were prepared from dioleoyphosphatidylcholine (DOPC), dipalmitoyphosphatidylcholine (DPPC) and cholesterol (Chol), and subsequently mixed to form solutions with 5.33:2.66:2, 2.665.3:3:2 and 4.8:3.2:2 DOPC:DPPC:Chol molar lipid ratios. The fluorescent dye N-(lyssamine rhodamine B sulphonyl)-dipalmitoylphosphatidylethanolamine (Rh-DPPE) was added to the mixture (0.1 mol %). A small volume (12 μl) of the lipid solution was spread on the conductive surfaces of two glass plates coated with indium tin oxide. The latter were kept at around 60°C and under vacuum for approximately 2 h to remove all traces of the organic solvent. The two glasses were placed with their conductive sides facing each other and separated by a 2 mmthick Teflon frame to form a chamber, which was sealed with silicon grease. The chamber was then gently filled with preheated sucrose solution (0.01 to 0.1 M) and placed in an oven at 60°C. The glass plates were connected to a function generator and an alternating current (700 mV, 10 Hz frequency) was applied for one hour. Vesicles with an average diameter of 50 μm and a large polydispersity were observed after about 2 hours. Then, a 5 Hz current of 2 V was applied for another hour to detach the vesicles from the glass surfaces. The vesicle solution was let to cool slowly, removed from the electroswelling chamber and diluted 40 times into an isotonic glucose solution. The osmolarities of the sucrose and glucose solutions were measured with cryoscopic osmometer Osmomat 030 (Gonotec, Berlin, Germany) and carefully matched to avoid an osmotic pressure difference. The conductivity of the glucose solution was adjusted by adding NaCl solution and measured with conductivity meter SevenEasy (Mettler Toledo, Greifensee, Switzerland). Approximately 1 ml of the obtained vesicle solution was placed in a chamber for electromanipulation purchased from Eppendorf (Hamburg, Germany). Because of the differences in density and refractive index between the sucrose and glucose solutions, the vesicles were stabilized by gravity at the bottom of the chamber. At room temperature, lipid membranes with the above composition are in the two-phase coexistence region. The vesicles exhibit liquid ordered (lo) and liquid disordered (ld) domains. Rh-DPPE is known to partition in the ld phase. After equilibration, the vesicles typically contained one lo domain and one ld domain. We preheated them to obtain a larger number of smaller domains before subjecting them to electric fields.