The overall goal of this protocol is to quantitatively assess the effect of the high salinity environments and transmembrane solution assymetry found in physiological conditions on membrane phase states. Solutions inside cell and outside cells are very different. Our methods help understand the effect of the solution assymetry on the membrane properties and phase behavior.
The main advantage of our approach is that vesicles can be prepared at physiological buffer conditions and the microfluidic device can be used to create asymmetric buffer conditions. To begin the procedure, roughen one side of a PTFE plate with fine sandpaper. Wash the plate, a 15 milliliter glass vial, and a sealable glass container with commercial dish-washing detergent, ethanol, and chloroform.
Use a 25 microliter glass syringe to deposit 10 to 15 microliters of lipid stock in chloroform onto the rough side of the clean, dry PTFE plate. Spread the lipid stock with the syringe needle to create a uniform lipid film. Use tweezers to transfer the plate to the 15 milliliter vial.
Heat the plate at 60 degrees Celsius and desiccate for two hours to evaporate the solvent. Then, add deionized water to a glass container to a height of about one centimeter. Set the vial with the plate in the container, ensuring that it remains stable and upright and seal the container.
Allow the lipid bilayer to pre-swell at 60 degrees Celsius for four hours. Then, remove the vial from the container. Draw five milliliters of swelling solution into a plastic syringe and attach a 0.45 micrometer filter to the syringe tip.
Add the filtered swelling solution to the vial to hydrate the lipid film. Cap the vial and seal the vial with plastic paraffin film. Store the vial at 60 degrees Celsius overnight to obtain the GUV aggregate.
Cool the vesicles to room temperature within an hour of removal from heat. Cut off the end of a 200 microliter plastic micro-pipette tip, so that the GUV aggregate can be drawn into the tip. Use the trimmed pipette tip to transfer the aggregate and 50 microliters of swelling solution to a clean, dry two milliliter vial.
Add to the vial 950 microliters of fresh swelling solution or an isotonic solution to attain symmetric or asymmetric solution conditions respectively. Pipette the mixture up and down and then gently invert the vial several times to resuspend the GUVs. To begin the assessment, place an aliquot of GUV suspension in a silicone isolator in a microscope slide.
Seal the chamber with a coverslip. Allow the sample to reequilibrate for five minutes. Then, place the slide on the sample stage of a fluorescence microscope and acquire images of at least 30 GUVs per batch.
Inspect the images to determine the phase states of the image GUVs. GUVs in a single liquid phase state will be spherical and smooth with a fluorescent label homogeneously distributed throughout the membrane. GUVs in a liquid ordered and disordered two-phase state will be spherical with a fluorescent label partitioned in domains with smooth boundaries within the membrane.
Verify that the domains are free to diffuse on the vesicle surfaces and can coalesce. GUVs in a two-phase or three-phase solid liquid phase state will be rounded with some angular boundaries. Liquid domains on a solid background should not display diffusion, but solid domains should freely diffuse on a liquid background.
Once the phase state of each image GUV has been identified, determine the dominant phase state for the batch. Repeat the process with independent samples if the dominant phase state is a narrow majority. Before using the microfluidic device, prepare at least five milliliters each of sucrose and salt swelling solutions.
Filter each solution through a 0.45 micrometer filter. Next, trim the ends of 200 microliter plastic pipette tips and place a pipette tip in each channel of the device. Add to the device reservoir 100 microliters of fresh filtered swelling solution of the same type used to prepare the GUVs.
Add five microliters of the swelling solution to each pipette tip. Centrifuge the device at 900 times G for 10 minutes to fill the device channels. Then, fill a one milliliter glass syringe with the same filtered swelling solution.
Connect a length of tubing to the syringe. Push the swelling solution through the tubing until the plunger is fully depressed and the tubing is filled with solution. Then, connect the tubing to the outlet channel and mount the syringe on a syringe pump.
Next, connect the pressure control unit to the microfluidic channel inlets. Ensure that the valves are closed and then set the pressure to three bars. Place the microfluidic device on the sample stage of an inverted confocal microscope.
Use a pipette to remove the swelling solution from the device reservoir until only 25 to 50 microliters of solution remain. Add 150 microliters of the GUV suspension to the reservoir and mix the suspension with gentle pipetting. Run the syringe pump in withdrawal mode at 10 microliters per minute for 20 minutes or until more than 90%of the device traps are occupied.
Then, open the pressure control unit valves to close the ring valves in the microfluidic device. Stop the syringe pump and allow the device to sit for one hour before acquiring confocal images of the GUVs in the device. Next, use a pipette to remove the GUV suspension from the reservoir to leave only 25 to 50 microliters.
Add to the reservoir 150 microliters of the other type of filtered swelling solution and gently pipette the mixture up and down. Repeat this process at least five times to completely replace the solution in the reservoir. Run the syringe pump in withdraw mode at 10 microliters per minute for 10 minutes to replace the solution in the microchannels.
Then, decrease the flow rate to one microliter per minute. Close the pressure control unit valves for two seconds and then open the valves and stop the syringe pump. Allow the device to sit for one hour before recording confocal images.
To begin preparing the temperature control chamber, first attach barbed tube fittings to an aluminum heat flow assembly with two millimeter thick coverslip windows. Attach a rubber spacer to one of the coverslips to form the observation chamber. Load 100 microliters of the GUV suspension into the observation chamber and seal the chamber with a coverslip.
Slowly invert the assembly. Connect the aluminum chamber to an external water bath. Mount the assembly on a microscope sample stage.
Set the external water bath to the lowest desired temperature and allow the system to equilibrate for 10 to 15 minutes. Acquire images of the GUVs and determine the phase states at various temperatures. GUVs were prepared from varying ratios of DOPG and egg sphingomyelin and cholesterol in symmetric sucrose or salt solutions.
Their dominant phase states were determined by fluorescence microscopy. Selected compositions were then transferred to asymmetric conditions by dilution to evaluate the effective solution conditions on the phase behavior. To further investigate GUV phase behavior in asymmetric solutions, GUVs were trapped in a microfluidic device to allow complete exchange of the external solution.
The trapped GUVs were evaluated by confocal microscopy before and after the fluidic exchange. The fraction of homogeneous to phase separated GUVs was monitored over a range of temperatures to evaluate the effective temperature on GUV phase behavior. A sigmoidal pattern was observed in the data, which were then fit to a Boltzmann model.
The temperature at which the fraction of phase-separated GUVs was 0.5 was determined from the fitted curve. Although this methods can provide insight into the membrane phase behavior, the applications can also be used for other experiments, such as to study the interactions between membranes in biomolecules at physiological conditions.
