Overview¶
Dye Liposomes encapsulate HPTS dye in Base Membrane and use a simple, glucose outer solution. Dye Liposomes are a fast debugging tool and positive control for liposome encapsulation and microscopy. This protocol is adapted from the Build a Cell liposome kit (Fujii et al., 2014).
Reference Composition¶
Inner Solution
Membrane
Outer Solution
| Component | Stock concentration | Final concentration | Volume for one reaction (µL) |
|---|---|---|---|
| HPTS | 4 mM | 0.2 mM | 5 |
| Optiprep | 1.32 mg/µL | 0.043 mg/µL | 1.33 |
| Water | 23.67 | ||
| Total volume (µL) | 30 |
| Component | Target Percentage (%) | Molecular Weight (g/mol) | Stock concentration (mg/mL) | Volume to add (µL) |
|---|---|---|---|---|
| POPC | 70 | 760.076 | 25 | 162.17 |
| Cholesterol | 29.95 | 386.654 | 50 | 17.65 |
| Liss-Rhod PE | 0.05 | 1301.71 | 1 | 4.96 |
See Base Membrane for the full membrane spec.
| Component | Concentration |
|---|---|
| Glucose | 800 mM |
Expected Behavior¶
Liposomes are visible in the green channel (interior, HPTS, 480 nm ex / 520 nm em) and in the red channel (membrane, Liss-Rhodamine-PE, 540 nm ex / 580 nm em) under fluorescence microscopy.
Process¶
Dye Liposomes are assembled and encapsulated using Encapsulation: Phase Transfer.
Materials and Equipment¶
| Name | Category | Product | Manufacturer | Part # | Price | Storage | Link |
|---|---|---|---|---|---|---|---|
| HPTS | Reagent | HPTS (Pyranine) | Thermo Scientific | L11252-06 | $92.60 | RT | link |
| HEPES pH 7.6 | Chemical | HEPES, Free Acid | Fisher BioReagents | NC1584172 | $85.90 | RT | link |
| Optiprep | Reagent | OptiPrep™ | STEMCELL Technologies | 07820 | $289.00 | RT | link |
| POPC | Lipid | 16:0-18:1 PC 25 mg/mL | Avanti Lipids | A80557 | $435.00 | -20 °C | link |
| Liss-Rhod PE | Lipid | 18:0 Liss Rhod PE 1 mg/mL | Avanti Lipids | A81179 | $273.47 | -20 °C | link |
| Glucose | Chemical | D-(+)-Glucose, 99% | Thermo Scientific | A16828-36 | $41.65 | RT | link |
| Mineral oil | Chemical | Mineral oil, mixed weight | Thermo Scientific | AC415080010 | $53.40 | RT | link |
| Glass syringe 250 µL | Equipment | Hamilton glass syringe | Hamilton | 14-815-238 | $150.15 | RT | link |
| 18-well imaging slide | Consumable | µ-Slide 18 Well glass bottom | ibidi | 80826 | $178.00 | RT | link |
- Fujii, S., Matsuura, T., Sunami, T., Nishikawa, T., Kazuta, Y., & Yomo, T. (2014). Liposome display for in vitro selection and evolution of membrane proteins. Nature Protocols, 9(7), 1578–1591. 10.1038/nprot.2014.107