diff --git a/Adapt_BF.JPG b/Adapt_BF.JPG new file mode 100644 index 0000000..80c4053 Binary files /dev/null and b/Adapt_BF.JPG differ diff --git a/Adapt_GFP.JPG b/Adapt_GFP.JPG new file mode 100644 index 0000000..69b0cac Binary files /dev/null and b/Adapt_GFP.JPG differ diff --git a/Adapt_Rod.JPG b/Adapt_Rod.JPG new file mode 100644 index 0000000..d0c0479 Binary files /dev/null and b/Adapt_Rod.JPG differ diff --git a/Adapt_overlay.JPG b/Adapt_overlay.JPG new file mode 100644 index 0000000..2e22cee Binary files /dev/null and b/Adapt_overlay.JPG differ diff --git a/Step0_LipidFilm.jpg b/Step0_LipidFilm.jpg new file mode 100644 index 0000000..0025d09 Binary files /dev/null and b/Step0_LipidFilm.jpg differ diff --git a/Step1_ResuspendLipidFilm.jpg b/Step1_ResuspendLipidFilm.jpg new file mode 100644 index 0000000..1911c56 Binary files /dev/null and b/Step1_ResuspendLipidFilm.jpg differ diff --git a/Step2_CentrifugeLayer.jpg b/Step2_CentrifugeLayer.jpg new file mode 100644 index 0000000..6fee2f7 Binary files /dev/null and b/Step2_CentrifugeLayer.jpg differ diff --git a/Step2_FinalVesicles.jpg b/Step2_FinalVesicles.jpg new file mode 100644 index 0000000..3c2b088 Binary files /dev/null and b/Step2_FinalVesicles.jpg differ diff --git a/Step2_LipidEmulsion.jpg b/Step2_LipidEmulsion.jpg new file mode 100644 index 0000000..c400561 Binary files /dev/null and b/Step2_LipidEmulsion.jpg differ diff --git a/Step2_WashVesicles.jpg b/Step2_WashVesicles.jpg new file mode 100644 index 0000000..77e8fe2 Binary files /dev/null and b/Step2_WashVesicles.jpg differ diff --git a/protocol-list.md b/protocol-list.md deleted file mode 100644 index 58ca290..0000000 --- a/protocol-list.md +++ /dev/null @@ -1,104 +0,0 @@ -# List of the common liposome protocols for the encapsulation of TX-TL mix - -## Principles -The most common liposomes protocols for encapsulating TX-TL mixtures are based on two strategies: -- hydration of dry lipids -- transformation of lipid-covered water-in-oil (w/o) droplets into liposomes (aka: droplet transfer / emulsion inversion) - -The first strategy leads to samples composed only by lipids and the aqueous phase used for hydration. The second one leads to samples which can contain also traces of oil either as oil droplets, either as oil solubilized in the liposome membrane. - -*Note 1*: by "oil" we mean an apolar solvent (generally a hydrocarbon, like dodecane or squalene, or a mixture of hydrocarbons, like the "mineral oil") - -**Hydration of dry lipids** - -We can distringuish three variants to this somehow "classical" preparation method. -1. hydration of a dry lipid film stratified inside a glass vial -2. hydration of a dry lipid film stratified on the surface of glass beads (diam. 0.2-2 mm) -3. hydration of freeze-dried liposomes - -The first two variants lead to liposome from multi-layer lipid lamellae deposited over the glass surface of a glass vial or of glass beads. To prepare the film, just let a chloroform solution of lipids evaporate. The third variant starts from freeze-dried liposomes. To prepare freeze-dried liposomes (sometimes referred to as "liposome cake"), liposomes must be firstly prepared by another method (e.g., hydration of lipid film), possibly extruded, then freeze-dried. - -In these three variants, liposomes are formed just by adding the aqueous solution to the lipids. To have large vesicles (possibly giant one), minimal shear forces should be applied. After preparation, the inner and the outer solution are the same, so that there are no osmotic issues. If liposomes are manipulated after formation, the outer solution should be kept isotonic with the inner one. - -**Droplet transfer** - -This is a "novel" preparation method. Firstly, lipid-stabilized w/o droplets are formed by simple emulsification of a small amount of aqueous phase in a lipid-containing oil, next the droplets are allowed to pass through a flat oil/water interface, also stabilized by lipids, to acquire the second lipid layer, and form liposomes. - -The method works well if the inner solution (that goes in the liposome lumen) and the outer solution are *isotonic* but have *different densities*. At this aim, sucrose and glucose are generally added to the inner and outer solution, respectively. Inner and outer solutions can have different buffer and different composition (e.g., TX-TL mix inside, PBS outside), provided that the two solutions are isotonic. - -## Some of the publications describing the above mentioned methods - -| Year | Reference | Size (diam.) | Comments | -| ---- | --------- | ----------- | ---------- | -| 1999 | [Oberholzer et al, Biochem Biophys Res Commun, 1999] | LUVs, 0.4 um | Hydration of lipid film in a vial| -| 2004 | [Ishikawa et al, FEBS Letters, 2004] |not given, probably few um | Rehydration of liposome "cake" | -| 2012 | [Danelon et al, Angew. Chem. Int. Ed, 2012] | <1-20 um | Hydration of lipid films on glass beads | -| 2014 | [Fujii et al, Nature Protocols, 2014] | 1-6 um | Droplet transfer | -| 2015 | [Ishiwata et al, Nature Protocols Exchange, 2015] | 1-10 um | Droplet transfer | - -## Short summaries and links - -### Oberholzer et al, Biochem Biophys Res Commun, 1999 - -This is the very first report of ribosomal polypeptide synthesis inside liposomes. The liposome formation follows the hydration of lipid film stratified inside a vial, followed by freeze-thaw cycles, then extrusion [Oberholzer et al, Biochem Biophys Res Commun, -1999](https://www.sciencedirect.com/science/article/pii/S0006291X99904047) -(note that the extrusion can be skipped). Additional work in this direction is descirbed in [Yu et al, J Biosci Bioeng, -2001](https://www.sciencedirect.com/science/article/pii/S1389172301803224) (the very first report on GFP synthesis inside liposomes). - -### Ishikawa et al, FEBS Letters, 2004 - -The preparation of liposomes is based on the hydration of freeze-dried extruded liposomes [Ishikawa et al, FEBS Letters, -2004](https://www.sciencedirect.com/science/article/pii/S0014579304011743), -based on a [previous -paper](https://www.sciencedirect.com/science/article/pii/S0168365999000474). The advantage of hydrating freeze-dried liposomes -rather then lipid films lies in the higher entrapment rate. - -### Danelon et al, Angew. Chem. Int. Ed, 2012 - -A method for hydration of a lipid film stratified over 0.2 mm glass beads has -been described by [Danelon et al, Angew. Chem. Int. Ed, -2012](https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201107123); -similar experiments (not referred to TX-TL reactions, and using 2 mm glass beads) can be found in an [MDPI Life -paper](http://www.mdpi.com/2075-1729/5/1/969). - -### Spencer et al, JoVE, 2013 - -This paper presents an approach similar to the above-mentioned [Ishikawa et al, FEBS Letters, -2004], and monitoring of protein production. A [JoVE paper and video](https://www.jove.com/video/51304/the-encapsulation-cell-free-transcription-translation-machinery) is available, based on the paper by [Sunami et al, Methods Mol. Biol., 2010](https://link.springer.com/protocol/10.1007%2F978-1-60327-331-2_20). - -### Fujii et al, Nature Protocols, 2014 - -This paper describes step-by-step, with explanatory pictures, the droplet transfer method. Yomo and collaborators have -used this method in dozens of publications. The protocol is available in -[Fujii et al, 2014](https://www.nature.com/articles/nprot.2014.107). -In the following [JoVE article](https://www.jove.com/video/55282) a similar method is applied for encapsulating microspheres in liposomes. A detailed protocol is also available in the [pdf companion article](https://www.jove.com/pdf/55282/jove-protocol-55282-preparation-giant-vesicles-encapsulating-microspheres-centrifugation) - -### Miyazaki et al, Nature Protocols Exchange, 2015 - -This is another description of the droplet transfer method, for the *in vitro* reconstitution of biological processes in cellular compartments. Here the [protocol](https://www.nature.com/protocolexchange/protocols/3815#/related-articles). - -A modified version of this protocol can be found at [OpenWetWare](https://openwetware.org/wiki/Preparation_of_cell-sized_water-in-oil_droplets) The protocol was used by Malin Jonsson, Miroslav Gasparek (summer 2017 interns), and Zoila Jurado (PhD student at Caltech) to express the GFP in the liposomes in the summer of 2017 in the Murray Lab at Caltech. - -Aaron Engelhart (University of Minnesota) -Richard Murray (Caltech) -Jan Gregrowicz -Zoila JURADO (Caltech) -Akshay Maheshwari (Stanford) -Neha (Northwestern) -Milena Popovic (Blue Marble Space) -Kazhito Tabata -Joseph Heili, University of Minnesota -Paola - -## Further readings - -To know more about general liposomes protocols: - -- [Szoka, F.; Papahadjopoulos, D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu. Rev. Biophys. Bioeng. 1980, 9, 467–508](https://www.annualreviews.org/doi/abs/10.1146/annurev.bb.09.060180.002343) -- [New, R. R. C. Liposomes. A practical approach.; 1st Edition.; IRL Press at Oxford University Press: Oxford, 1990](https://www.amazon.com/Liposomes-Practical-Approach/dp/0199630771) -- [Walde, P. Preparation of Vesicles (Liposomes) ASP Encyclopedia of Nanoscience and Nanotechnology (2003), Ed. H. S. Nalwa, Vol. 9, 43-79](http://www.aspbs.com/enna-z.html) -- [Walde, P.; Cosentino, K.; Hengel, H.; Stano, P. Giant Vesicles: Preparations and Applications. ChemBioChem 2010, 11, 848-865](https://onlinelibrary.wiley.com/doi/abs/10.1002/cbic.201000010) - - - - diff --git a/txtl-liposome_water-in-oil.md b/txtl-liposome_water-in-oil.md index 644e321..384b6f4 100644 --- a/txtl-liposome_water-in-oil.md +++ b/txtl-liposome_water-in-oil.md @@ -1,210 +1,222 @@ -# TX-TL Liposome Using Water-in-Oil Emulsion - -Version 0.1.0, 8 August 2018 - -Contributors: -Aaron Engelhart (University of Minnesota), -Jan Gregrowicz (Caltech), -Joseph Heili (University of Minnesota), -Zoila Jurado (Caltech), -Neha Kama (Northwestern), -Akshay Maheshwari (Stanford), -Richard Murray (Caltech), -Milena Popovic (Blue Marble Space), -Pasquale Stano (University of Salento), -Kazhito Tabata (University of Tokyo), -Paola Torre (University of Pennsylvania). - -## Overview and Materials - -This protocol describes how to create liposomes that contain TX-TL -inside of a lipid-based container. The liposomes created by these -protocols are between 1 and 10 um in diameter and should be usable -with an cell-free protein expression system. - -The preparation of liposomes using w/o emulsions as template requires -four steps: - -### Step 0: Deposition of a thin lipid film in a glass vial - -#### Materials: - -* Glass vials (any will do, but we use [2mL Fisherbrand Class B Clear - Glass Threaded vials: cat #: - 03-339-21A](https://www.fishersci.com/shop/products/fisherbrand-class-b-clear-glass-threaded-vials-with-closures-packaged-separately-12/p-204738) - -* POPC in chloroform: [https://avantilipids.com/product/850457](https://avantilipids.com/product/850457) - -* Liss-Rhod-PE: - [https://avantilipids.com/product/810150](https://avantilipids.com/product/810150) - -* Cholesterol in chloroform (25 mg/ml): https://avantilipids.com/product/700000 - -* Glass syringes of various sizes (10 uL, 50 uL, 250 uL, and 1 mL are - suggested ex. Hamilton gastight cat #: 14-815-238, but any will do) - for use with lipids and chloroform - [https://www.hamiltoncompany.com/products/syringes-and-needles/general-syringes/gastight-syringes](https://www.hamiltoncompany.com/products/syringes-and-needles/general-syringes/gastight-syringes) - -### Step 1: Preparation of the oil containing lipids - -#### Materials: - -* Mineral oil: [https://us.vwr.com/store/product/7422728/mineral-oil-light-white-high-purity-grade](https://us.vwr.com/store/product/7422728/mineral-oil-light-white-high-purity-grade) - -### Step 2: Self-assembly of the Liposomes by centrifugation - -#### Materials: - -* 20 uM of HPTS stock solution: ([https://www.thermofisher.com/order/catalog/product/H348](https://www.thermofisher.com/order/catalog/product/H348)) - -* 100 mM HEPES, 200 mM glucose pH 8 - -* 100 mM HEPES + 250 mM glucose, pH 8 - -* 100 mM HEPES, 200 mM sucrose pH 8 - -### Step 3: Microscopy Visualization - -#### Materials: - -* Frame Seals: [https://www.thermofisher.com/order/catalog/product/S24736](https://www.thermofisher.com/order/catalog/product/S24736) - -* Glass slides - -## Step 0: Deposition of a thin lipid film in a glass vials - -The first step in the protocol is to generate the lipid film required -to to form the lipid-in-mineral oil solution. Makes 6 samples with -accurate measurement. Each vial will have ~ 15 mg of lipid with 0.1 +# TX-TL Liposome Using Water-in-Oil Emulsion + +Version 0.1.0, 8 August 2018 + +Contributors: +Aaron Engelhart (University of Minnesota), +Jan Gregrowicz (Caltech), +Joseph Heili (University of Minnesota), +Zoila Jurado (Caltech), +Neha Kama (Northwestern), +Akshay Maheshwari (Stanford), +Richard Murray (Caltech), +Milena Popovic (Blue Marble Space), +Pasquale Stano (University of Salento), +Kazhito Tabata (University of Tokyo), +Paola Torre (University of Pennsylvania). + +## Overview and Materials + +This protocol describes how to create liposomes that contain TX-TL +inside of a lipid-based container. The liposomes created by these +protocols are between 1 and 10 um in diameter and should be usable +with an cell-free protein expression system. + +The preparation of liposomes using w/o emulsions as template requires +four steps: + +### Step 0: Deposition of a thin lipid film in a glass vial + +#### Materials: + +* Glass vials (any will do, but we use [2mL Fisherbrand Class B Clear + Glass Threaded vials: cat #: + 03-339-21A](https://www.fishersci.com/shop/products/fisherbrand-class-b-clear-glass-threaded-vials-with-closures-packaged-separately-12/p-204738) + +* POPC in chloroform: [https://avantilipids.com/product/850457](https://avantilipids.com/product/850457) + +* Liss-Rhod-PE: + [https://avantilipids.com/product/810150](https://avantilipids.com/product/810150) + +* Cholesterol in chloroform (25 mg/ml): https://avantilipids.com/product/700000 + +* Glass syringes of various sizes (10 uL, 50 uL, 250 uL, and 1 mL are + suggested ex. Hamilton gastight cat #: 14-815-238, but any will do) + for use with lipids and chloroform + [https://www.hamiltoncompany.com/products/syringes-and-needles/general-syringes/gastight-syringes](https://www.hamiltoncompany.com/products/syringes-and-needles/general-syringes/gastight-syringes) + +### Step 1: Preparation of the oil containing lipids + +#### Materials: + +* Mineral oil: [https://us.vwr.com/store/product/7422728/mineral-oil-light-white-high-purity-grade](https://us.vwr.com/store/product/7422728/mineral-oil-light-white-high-purity-grade) + +### Step 2: Self-assembly of the Liposomes by centrifugation + +#### Materials: + +* 20 uM of HPTS stock solution: ([https://www.thermofisher.com/order/catalog/product/H348](https://www.thermofisher.com/order/catalog/product/H348)) + +* 100 mM HEPES, 200 mM glucose pH 8 + +* 100 mM HEPES + 250 mM glucose, pH 8 + +* 100 mM HEPES, 200 mM sucrose pH 8 + +### Step 3: Microscopy Visualization + +#### Materials: + +* Frame Seals: [https://www.thermofisher.com/order/catalog/product/S24736](https://www.thermofisher.com/order/catalog/product/S24736) + +* Glass slides + +## Step 0: Deposition of a thin lipid film in a glass vials + +The first step in the protocol is to generate the lipid film required +to to form the lipid-in-mineral oil solution. Makes 6 samples with +accurate measurement. Each vial will have ~ 15 mg of lipid with 0.1 mol % of 18:1 Lyss-Rho-PE. - -1. Create lipid master mix in a glass beaker. One of two methods may be used: - - * POPC/Lyss-Rhod-PE - - a. Add 4 mL (100 mg) of POPC in chloroform (25 mg/ml) - - b. Add 200 uL (0.2 mg) of Lyss-Rho-PE in chloroform (1 mg/mL) - - c. Swirl gently until all POPC is dissolved and color is homogeneous. - - * POPC/Cholesterol/Lyss-Rhod-PE - - a. Add 2.668 mL (66.7 mg) of POPC in chloroform (25 mg/ml) - - b. Add 1.332 mL (33.3 mg) of Cholesterol in chloroform (25 mg/ml) - - c. Add 200 uL (0.2 mg) of Lyss-Rho-PE in chloroform (1 mg/mL) - - d. Swirl gently until all POPC is dissolved and color is homogeneous. - -2. Aliquot 700 uL of POPC/Cholesterol/Lyss-Rhod-PE Chloroform solution -into 2 mL glass vials (6 vials total) - -3. Place uncapped vials in fume hood, loosely covered with aluminum -foil and allow to evacuate overnight in fume hood (~6-8 hours). - - * **Note:** The purpose of the aluminum foil cover is to protect - particles from contaminating the POPC/Cholesterol/Lyss-Rhod-PE - as it evaporates. - -4. Move vials to vacuum chamber and vacuum for an additional 2 hours. - -5. Store in -20 degC. - -**Note:** Remaining POPC in chloroform, cholesterol, and Lyss-Rho-PE in chloroform can be stored at -20 degC in the glass vial with PTFE caps and sealed parafilm. - -## Step 1: Preparation of the oil containing lipids - -1. Mix mineral oil by gentle inversion before use - -2. Place 0.5 mL of mineral oil into each of the vials. - -3. Incubate at 60 degC for 10 min - -4. Vortex for 10 mins - -5. Incubate vials for 3 hrs at 60 degC. - -6. Wrap up the top of the vials with aluminum foil and seal with parafilm. - -7. Sonicate in an heated water bath for 30 minutes,at 60 degC . - - a. **Note:** If a sonicator is unavailable then repeat 60 degC - incubation and vortexing until the lipid is completely dissolved. - -## Step 2: Self-assembly of the Liposomes by centrifugation - -1. Place 225 uL of centrifuge buffer (100 mM HEPES + 200 mM glucose, - pH 8) into a labeled eppendorf tube. - - a. **Note**: When TX-TL systems are used, the outer solution should - also contain the small molecular weight components of the TX-TL - system. - -2. Add in 30 uL of inner solution (100 mM HEPES, 200 mM sucrose pH 8, - and, if needed, 2 uM of a water-soluble fluorescent dye like HPTS - or calcein.) to 500 uL of suspended lipid in oil from Step - 1. Vortex for 30 s. Equilibrate for 10 min at 4 degC. - - * **Note**: When TX-TL systems are used, buffer is omitted. TX-TL - systems come with their own buffers. - -3. Add the emulsion on top of the 225 uL centrifuge buffer by -pipetting against the wall of the tube, wait at least 1 minute for the -interface to stabilize and flatten between the emulsion and buffer - -4. Centrifuge at 18000 rcf at 4 degC for 15 min - - * **Note:** If the top phase (oil) is clear, that suggests that - droplets have passed into the bottom buffer, becoming vesicles. - -5. Carefully remove as much mineral oil as possible with a - gel-loading-pipette-tip from the top. - - * **Note: **The goal is to remove as much oil up to the interface - without disturbing the buffer below. - -6. Place 225 uL of wash buffer (100 mM HEPES + 250 mM glucose, pH 8) - into a labeled eppendorf tube. - -7. Add the centrifuge buffer (where the vesicles should have formed) - to eppendorf tubes with wash buffer. Make sure to use a new tip to - avoid contamination. - - * **Note:** An alternative method is to open the eppendorf tube - and use a 21-gauge needle to punch a hole at the bottom of the - eppendorf tube. Remove the needle and the close the lid to allow - the buffer solution to drip out. - -8. Centrifuge at 12000 rcf at 4 degC for 5 min - -9. Transfer 225 uL from the bottom of the eppendorf tubes with wash - buffer into a new labeled eppendorf tube. - -## Step 3: Microscopy Visualization - -1. Use Frameseal or Spacer (20 mm D x 0.12 mm depth) to make a small - chamber on a microscope slide. - -2. Add 10 uL of the final solution to the chamber and seal with cover slip - -3. Wait 5 minutes before imaging (since the liposomes will float - everywhere and can be tricky to catch them). - -4. Observe on an inverted microscope. [Sample images] of what you should see: - -### Alternative method (neha): - -* Use a glass bottom chamber: - [https://www.thermofisher.com/order/catalog/product/154453](https://www.thermofisher.com/order/catalog/product/154453) - -* Block glass by adding 0.5 mL of a 1mg/mL BSA in PBS solution for 10 - minutes (alternatively block by adding 0.5 mL o[f - SuperBlock](https://www.thermofisher.com/order/catalog/product/37515) - for 5 min). The blocking step is important because phospholipid - vesicles will rupture to some degree on glass. - -* Rinse with PBS 3X. Add 0.5 mL of final buffer from above to - chamber. Add 10 uL of vesicle sample to chamber, let settle over a - few minutes. Image on scope with green and red channels. + +1. Create lipid master mix in a glass beaker. One of two methods may be used: + + * POPC/Lyss-Rhod-PE + + a. Add 4 mL (100 mg) of POPC in chloroform (25 mg/ml) + + b. Add 200 uL (0.2 mg) of Lyss-Rho-PE in chloroform (1 mg/mL) + + c. Swirl gently until all POPC is dissolved and color is homogeneous. + + * POPC/Cholesterol/Lyss-Rhod-PE + + a. Add 2.668 mL (66.7 mg) of POPC in chloroform (25 mg/ml) + + b. Add 1.332 mL (33.3 mg) of Cholesterol in chloroform (25 mg/ml) + + c. Add 200 uL (0.2 mg) of Lyss-Rho-PE in chloroform (1 mg/mL) + + d. Swirl gently until all POPC is dissolved and color is homogeneous. + +2. Aliquot 700 uL of POPC/Cholesterol/Lyss-Rhod-PE Chloroform solution +into 2 mL glass vials (6 vials total) + +3. Place uncapped vials in fume hood, loosely covered with aluminum +foil and allow to evacuate overnight in fume hood (~6-8 hours). + + * **Note:** The purpose of the aluminum foil cover is to protect + particles from contaminating the POPC/Cholesterol/Lyss-Rhod-PE + as it evaporates. + +4. Move vials to vacuum chamber and vacuum for an additional 2 hours. + +5. Store in -20 degC. + +![Lipid film preparation](https://github.com/zjuradoq/liposome-kit/blob/master/Step0_LipidFilm.jpg) + +**Note:** Remaining POPC in chloroform, cholesterol, and Lyss-Rho-PE in chloroform can be stored at -20 degC in the glass vial with PTFE caps and sealed parafilm. + +## Step 1: Preparation of the oil containing lipids + +1. Mix mineral oil by gentle inversion before use + +2. Place 0.5 mL of mineral oil into each of the vials. + +3. Incubate at 60 degC for 10 min + +4. Vortex for 10 mins + +5. Incubate vials for 3 hrs at 60 degC. + +6. Wrap up the top of the vials with aluminum foil and seal with parafilm. + +7. Sonicate in an heated water bath for 30 minutes,at 60 degC . + +![Lipid resuspended in 0.5 mL mineral oil](https://github.com/zjuradoq/liposome-kit/blob/master/Step1_ResuspendLipidFilm.jpg) + + a. **Note:** If a sonicator is unavailable then repeat 60 degC + incubation and vortexing until the lipid is completely dissolved. + +## Step 2: Self-assembly of the Liposomes by centrifugation + +1. Place 225 uL of centrifuge buffer (100 mM HEPES + 200 mM glucose, + pH 8) into a labeled eppendorf tube. + + a. **Note**: When TX-TL systems are used, the outer solution should + also contain the small molecular weight components of the TX-TL + system. + +2. Add in 30 uL of inner solution (100 mM HEPES, 200 mM sucrose pH 8, + and, if needed, 2 uM of a water-soluble fluorescent dye like HPTS + or calcein.) to 500 uL of suspended lipid in oil from Step + 1. Vortex for 30 s. Equilibrate for 10 min at 4 degC. + + ![Internal solution emulsion with mineral oil, monolayer vesicles.](https://github.com/zjuradoq/liposome-kit/blob/master/Step2_LipidEmulsion.jpg) + + * **Note**: When TX-TL systems are used, buffer is omitted. TX-TL + systems come with their own buffers. + +3. Add the emulsion on top of the 225 uL centrifuge buffer by +pipetting against the wall of the tube, wait at least 1 minute for the +interface to stabilize and flatten between the emulsion and buffer + +![Stablized emulsion and mineral oil interface, prior to creating of bilayer vesicles.](https://github.com/zjuradoq/liposome-kit/blob/master/Step2_CentrifugeLayer.jpg) + +4. Centrifuge at 16000 rcf at 4 degC for 8 min + + * **Note:** If the top phase (oil) is clear, that suggests that + droplets have passed into the bottom buffer, becoming vesicles. + +![Formation of bilayer vesicles, and phase separation of oil, free lipids, vesicles, and buffer solutions.](https://github.com/zjuradoq/liposome-kit/blob/master/Step2_WashVesicles.jpg) + +5. Carefully remove as much mineral oil as possible with a + gel-loading-pipette-tip from the top. + + * **Note: **The goal is to remove as much oil up to the interface + without disturbing the buffer below. + +6. Place 225 uL of wash buffer (100 mM HEPES + 250 mM glucose, pH 8) + into a labeled eppendorf tube. + +7. Add the centrifuge buffer (where the vesicles should have formed) + to eppendorf tubes with wash buffer. Make sure to use a new tip to + avoid contamination. + + * **Note:** An alternative method is to open the eppendorf tube + and use a 21-gauge needle to punch a hole at the bottom of the + eppendorf tube. Remove the needle and the close the lid to allow + the buffer solution to drip out. + +8. Centrifuge at 12000 rcf at 4 degC for 5 min + +9. Transfer 225 uL from the bottom of the eppendorf tubes with wash + buffer into a new labeled eppendorf tube. + +![Final solution of vesicles and buffer solution. Will be used in mircoscopy step.](https://github.com/zjuradoq/liposome-kit/blob/master/Step2_FinalVesicles.jpg) +## Step 3: Microscopy Visualization + +1. Use Frameseal or Spacer (20 mm D x 0.12 mm depth) to make a small + chamber on a microscope slide. + +2. Add 10 uL of the final solution to the chamber and seal with cover slip + +3. Wait 5 minutes before imaging (since the liposomes will float + everywhere and can be tricky to catch them). + +4. Observe on an inverted microscope. Potential vesicles of what you should see using 40X magnification: +![Bright-field, GFP, and RFP overlay from 40X mag images of potential vesicles](https://github.com/zjuradoq/liposome-kit/blob/master/Adapt_overlay.JPG) + +### Alternative method (neha): + +* Use a glass bottom chamber: + [https://www.thermofisher.com/order/catalog/product/154453](https://www.thermofisher.com/order/catalog/product/154453) + +* Block glass by adding 0.5 mL of a 1mg/mL BSA in PBS solution for 10 + minutes (alternatively block by adding 0.5 mL o[f + SuperBlock](https://www.thermofisher.com/order/catalog/product/37515) + for 5 min). The blocking step is important because phospholipid + vesicles will rupture to some degree on glass. + +* Rinse with PBS 3X. Add 0.5 mL of final buffer from above to + chamber. Add 10 uL of vesicle sample to chamber, let settle over a + few minutes. Image on scope with green and red channels.