The overall goal of this water-in-oil emulsion centrifugation method is to easily prepare a thin layer giant vesicle. This method can help answer key questions in the synthetic biology field such as the creation of an artificial cell based on a giant vesicle. To begin this procedure, prepare a 25 millimolar stock solution of DOPC and a zero point two millimolar stock solution of Texas Red DHPE in chloroform.
Next, form a lipid film on the inside surface of a five milliliter glass vial by evaporating a mixture of the DOPC and Texas Red DHPE stock solutions under flowing nitrogen gas. After incubating the film under reduced pressure overnight, add one milliliter of liquid paraffin to the vial. Wrap the vial in aluminum foil and incubate the mixture at 80 degrees Celsius overnight.
In a one point five milliliter lidded microtube mix 237 point five microliters of one micrometer nonfluorescent microspheres and 12 point five microliters of one micrometer fluorescent microspheres. Now add 64 milligrams of sucrose followed by 125 microliters of one molar Tris buffered solution and 875 microliters of deionized water to the microtube. Vortex the mixture for 30 seconds, then sonicate for 10 minutes.
After preparation of 10 milliliters of a Tris buffered solution, place one milliliter in a one point five milliliter lidded microtube. Once the solution has been vortexed and sonicated mix one milliliter of the oil solution with 300 microliters of the inner aqueous solution in a one point five milliliter microtube. Emulsify the two components in the microtube by using a mechanical homoginizer operated at 10, 000 rpm for two minutes at room temperature.
Next, gently layer 300 microliters of the water-in-oil emulsion on the upper surface of one milliliter of the outer aqueous solution at four degrees Celsius in a one point five milliliter lidded microtube. Immediately after chilling the microtube for 10 minutes centrifuge the mixture at 18, 000 times G for 30 minutes. When finished, obtain the precipitated giant vesicles or GVs by piercing the bottom of the microtube with a push pin and collecting one droplet in a sterilized one point five milliliter microtube.
Place an adhesive incubation chamber for NC two polymerase chain reaction and hybridization on top of a microscope cover glass. Using a micropipette, deposit 25 microliters of the diluted precipated GVs on the specimen area and immediately place a zero point one five millimeter thick cover glass on top of the incubation chamber. After recording microscopy images of the vesicles, conduct fluorescence microscopy by first inserting UFBNA and UFMCHE fluorescence mirror units into the microscope.
Then fit the units with excitation filters and emission filters for the analysis. The most important determinant of the success of the water-in-oil method is that the specific gravity of the inner aqueous solution must be larger than that of the outer aqueous solution so that the GVs will precipitate during centrifugation. Differential interference microscopy and fluorescence microscopy of the GVs without microspheres and with microspheres confirmed that GVs with low lamellarity were formed.
Of the 160 GVs obtained, 55 encapsulated microspheres and 105 were empty giving a 34%ratio of encapsulation. The volume fraction of microspheres in the GVs was estimated to be about 11 plus or minus three volume percent and the precision of the calculated volume fraction was 10 to 30%Encapsulation of other materials into 100 mole percent DOPC GVs using the same outer aqueous solution and the same protocol was successful as shown by differential interference microscopy and fluorescence microscopy. Following this procedure, other methods like flow cytometry can be performed in order to answer additional questions like elucidation of the modeled cell.
After watching this video, you should have a good understanding of how to prepare giant vesicles with encapsulated materials.
