Buffer-in-Buffer Liposomes

Printable Version

Nucleus Buffer-in-Buffer Liposomes Protocol.pdf

<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Overview

</aside>

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	Prepare stock buffers and lipids	1 h	4 h	Buffers and lipids may be prepared in advance and used for experiments on subsequent days.
2	Encapsulate liposomes	1 h	1 h
3	Measure and image	1 h	1 h

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

</aside>

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]

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 1: Prepare Stock Buffers and Lipids

</aside>

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 0.0021 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.

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	0.0021	1.0

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 2: Encapsulate inner solution into Liposomes

</aside>