Printable Version
Overview
Buffer-in-Buffer liposomes are primarily a teaching tool to gain familiarity with the process of making liposomes for synthetic cell building.
In this protocol, you will make necessary precursors to creating liposomes and encapsulate a simple fluorescent buffer within liposomes. The inner solution enclosed by the liposome will contain a sucrose solution labelled with a fluorescent dye. The outer solution will consist of a glucose solution.
When working, these liposomes should be easily visualized using green-channel epifluorescence microscopy.
There are three key stages to making Buffer-in-Buffer liposomes:
Step | Process | Hands-on Time | Total Time | Notes |
1 | 1 h | 4 h | Buffers and lipids may be prepared in advance and used for experiments on subsequent days. | |
2 | 1 h | 1 h | ||
3 | 1 h | 1 h |
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Link |
Buffers | |||||
Glucose | D-(+)-Glucose, 99% | Thermo Scientific | A16828-36 | $41.65 | [link] |
Sucrose | Sucrose, 99% | Thermo Scientific | A15583-36 | $41.65 | [link] |
HPTS (Pyranine) | Pyranine | Thermo Scientific | L11252-06 | $51.65 | [link] |
HEPES (pH 7.6) | HEPES Buffer | Fisher Scientific | NC1584172 | $108.86 | [link] |
Lipids | |||||
POPC | 16:0-18:1 PC 25 mg/mL | Avanti Lipids | 850457C-500mg | $435.00 | [link] |
Liss-Rhod-PE | 18:0 Liss Rhod PE 1 mg/mL | Avanti Lipids | 810179P-1mg | $273.47 | [link] |
Mineral Oil | Mineral oil, mixed weight | Thermo Scientific | AC415080010 | $53.40 | [link] |
Glass Syringe 250 uL | Hamilton | 14-815-238 | $150.15 | [link] |
Step 1: Prepare Stock Buffers and Lipids
Stock buffers may be prepared ahead of time, and stored for months. Lipids may also be prepared in advance and stored at 4C for at least 1 month. If you are making buffers and lipids at the same time, begin with the lipids so that they can evaporate while the buffers are being prepared.
Prepare lipids in mineral oil
Clean glass syringes.
Pour a small amount of 95% ethanol into a glass container (e.g. a 10 mL beaker).
Assemble the glass syringe and prime it by drawing ethanol into the glass syringe, then empty into a waste bottle.
Weight 2.67 g (or pipette 3.25mL) mineral oil into a glass test tube or beaker
Add the lipids to the mineral oil using the appropriate glass syringe:
Add 250 uL 25 mg/mL POPC.
Rinse the syringe with ethanol.
Add 10 uL 1 mg/mL Liss Rhod PE.
Rinse the syringe with ethanol.
Evaporate the chloroform from the lipid–oil mixture:
Place lipid beaker in a 55°C dry bath in a fume hood.
(optional) Shield with aluminum foil to protect from light.
Evaporate uncovered for 3 h.
Clean syringes and store with plunger removed
Let the sample cool to room temperature in the fume hood.
If lipids settle towards the bottom, stir them using a 10 uL pipette tip.
Aliquot the lipid–oil mixture into 1.5 mL aliquots, in glass jars.
The lipid–oil mixture can be stored for a week or more at room temperature, or up to one month at 4C.
Invert gently 3 times before use. Make sure the solution is not cloudy.
Prepare stock solutions
Buffer | Target Concentration (M) | MW (kDa) | Weight (g) | Final Volume (mL) |
3M Glucose Stock | 3.0 | 180.16 | 5.40 | 10.0 |
2M Sucrose Stock | 2.0 | 342.3 | 10.27 | 15.0 |
4mM HPTS Stock | 0.004 | 524.37 | 2.1 | 1.0 |
Make 3M glucose stock solution:
Combine 5.40 g glucose and 5.0 mL ddH2O in a 50 mL tube.
Heat for 15 s in a microwave and mix vigorously to dissolve material completely.
Add additional MilliQ water to achieve a final volume of 10 mL.
Filter sterilize while warm using a 0.22 um filter and store at -20 C.
Make 2M sucrose stock solution:
Combine 10.27 g sucrose and 5.0 mL ddH2O in a 50 mL tube.
Heat and mix vigorously to dissolve material completely.
Add additional ddH2O to achieve a final volume of 15 mL.
Filter sterilize using a 0.22 um filter and store at -20 C.
Make 4mM HPTS stock solution
Combine 2.1 mg HPTS and 1 mL ddH2O
Prepare outer buffer
Make a 800 mM glucose outer solution:
Add 1.6 mL 3M glucose stock solution to a 15 mL tube.
Add ddH2O water to a final volume of 6 mL.
Vortex vigorously to mix.
Prepare inner buffer
Make a 800 mM sucrose outer solution:
Add 0.6 mL ddH2O to a 1.5 mL microfuge tube.
Add 0.4 mL 2M sucrose stock solution to a final volume of 1 mL.
Vortex vigorously to mix.
Add dye and buffer
Add 95 uL to a 1.5 mL microfuge tube.
Add 5 uL 4mM HPTS stock solution
(optional) Add 1 uL 1M HEPES (pH 7.6) - HPTS is brighter at higher pH.
Step 2: Encapsulate inner solution into Liposomes
Set up a microfuge tube rack, with three 1.5 mL microfuge tubes per liposome encapsulation:
Label the three tubes:
E—oil emulsion
T—transfer
L—liposomes
‣
Add 150 uL of the lipid / oil mixture to the tube labelled E.
Add 150 uL of 800 mM glucose outer solution to the tubes labelled T and L.
Emulsify the mixture in the appropriate oil tube:
Add 30 uL inner solution to the E tube.
Mix the lipid–oil and inner solution by running the tube along a row of empty slots on the microfuge tube rack.
Run along the row 5–10 times, until the solution forms a stable emulsion and is an even milky color.
‣
Immediately layer each emulsion over the transfer solution.
Pipette 150 uL from tube E slowly down the side of the matching tube T.
‣
Centrifuge the T tube at 9000 g for 10 min at room temperature to pellet the liposomes:
Align the hinges of each microfuge tube towards the outside of the centrifuge rotor, so that the final pellet location will be indicated by the tube hinge.
‣
‣
Extract the liposomes from each T tube:
Remove the oil layer from the top of each T tube by gently pipetting with a 1000 uL pipette set to 150 uL.
Identify location of the liposome pellet at the corner of the tube where the side wall angles toward the bottom, directly below the hinge.
Gently extract liposomes by pipetting 50 uL of pellet and outer solution from beside the pellet location.
Add the 50 uL liposome sample to the matching liposome tube, L.
(optional) Repeat the previous step to reduce background fluorescence of HPTS by performing a second centrifugation.
‣
‣
Step 3: Measure Liposomes
Prepare a 18-well imaging slide for microscopy.
Pipette 20 uL of each liposome sample into a well on the slide.
Remaining liposomes may be stored at 4C.
Configure microscope:
Set magnification to 4x.
Set up imaging conditions for brightfield, GFP fluorescence (480 nm excitation, 520 nm emission), and Rhodamine fluorescence (540 nm excitation, 580 nm emission).
Place the slide on the microscope and prepare for imaging:
Find focus by focusing on the edge of a single well in brightfield.
Locate a well containing a sample of interest.
Move to imaging resolution:
Switch to rhodamine / red fluorescence imaging.
Find focus on liposomes just above the coverslip:
Back focus out until an entire field of liposomes appear to lose focus simultaneously (they are all sitting on the coverslip).
Slowly move back in until these liposomes are in sharp focus.
Move up to a higher magnification, and find focus again as described.
Repeat until you are at an imaging magnification of 20x or 40x.
Capture images of liposomes.
Background Protocols
Prepare lipids for use in encapsulation: 
Lipid Preparation
Lipid Preparation 
Resources and References
- Other Protocols
- The Nucleus protocol simplifies this previous work:The Build a Cell Phase-Transfer Liposome Protocol.
- Modified protocol from Miyazaki et al on OpenWetWare.
- Miyazaki protocol on Protocol Exchange
- Papers
- Fujii, S., Matsuura, T., Sunami, T. et al. Liposome display for in vitro selection and evolution of membrane proteins. Nat Protoc 9, 1578–1591 (2014) [link]
- Resources
Credits
- Developers
- Testers
