Nov 02, 2023
Version 2
Vesicle Fusion on SiO2 Substrates V.2
- 1Northeastern University
Protocol Citation: Nicole Voce, Paul Stevenson 2023. Vesicle Fusion on SiO2 Substrates. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3b4ylk5/v2Version created by Paul Stevenson
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 02, 2023
Last Modified: November 02, 2023
Protocol Integer ID: 90334
Keywords: supported lipid bilayer, membranes, lipid bilayer
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Abstract
This protocol outlines the steps to produce large area, uniform supported lipid bilayers on SiO2 substrates with or without patterned features.
Substrate cleaning
Substrate cleaning
- Clean plain SiO2 or TiO2-patterned substrates by sonicating them in a 1:1 IPA:Acetone mixture for ~2 minutes
- Dry with a steady stream of N2
Oxygen plasma clean the substrates for 2 minutes to remove any residual surface contamination
Note
For our Anatech SP-100 system, the RF power is set to 120 watts, the O2 flow rate to 15 ccm, and the pressure to 0.35-0.40 torr
Vesicle prep
Vesicle prep
Prepare a 1.2 mg/mL DLPC 0.5mol% Rho-PE solution (suitable for FRAP measurements and general fluorescence imaging) or a 0.3mg/mL DLPC 0.0025mol% Rho-PE solution (suitable for FCS measurements)
To prepare 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solutions:
- Measure out appropriate amount of DLPC (stored as powder) into a glass vial
Note
6 mg of DLPC for FRAP concentration; 1.55 mg of DLPC for FCS concentration
- Add appropriate amount of Rho-PE (stored as 1 mg/mL in chloroform) to DLPC
Note
60.6 μL of 1 mg/mL Rho-PE for FRAP concentration; 0.0781 μL of 1 mg/mL Rho-PE for FCS concentration
mol % = mols Rho-PE/(mols Rho-PE + mols DLPC)
For 1.2 mg/mL DLPC 0.5mol%:
- 0.005 = x/(x + 0.006g/621.85g/mol)
- Add 100-200 μL chloroform to vial
- Dry with N2 so the solution doesn't look glassy-- it should have a dull appearance
Glassy: FRAP solution before N2 dry
Past glassy: FRAP solution after N2 dry
- Leave in vacuum desiccator overnight to fully remove residual chloroform.
After drying, re-hydrate solution with phosphate buffered saline to achieve desired concentrations of 1.2 mg/mL or 0.3 mg/mL
Note
After adding phosphate buffered saline to the solution, shake or sonicate the glass vial(s) for ~30 seconds or until the dried DLPC/Rho-PE have been fully dissolved
Re-hydrated FRAP solution
Re-hydrated FCS solution
Extrude 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solution 21 times
Note
Equipment
Mini Extruder
NAME
Avanti Polar Lipids
BRAND
610000-1EA
SKU
LINK
Equipment
19mm Nuclepore™ Polycarbonate Track-Etched Membranes
NAME
Whatman
BRAND
10419504
SKU
LINK
Equipment
10mm Filter Supports
NAME
Whatman
BRAND
610014-1EA
SKU
LINK
To prepare the Mini Extruder, place filter supports in the middle of the rubber o-rings on the plastic cylinders. Deposit ~15 μL phosphate buffered saline on each support and use the pipette tip to move buffer around so each support is fully wet. Place the 100nm filter membrane on one of the supports, making sure it's secure with the phosphate buffered saline. Place the cylinder with the filter 3/4 of the way into the metal cylinder, place the other cylinder on top, sandwiching the membrane filter between the filter supports. Allow the cylinders to rest fully in the metal cylinder, then screw on the metal nut. Use one of the extruder syringes to measure out at least 300 μL of 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solution and stick the syringe into one end of the just-assembled filter apparatus. Stick the other extruder syringe into the other end of the filter apparatus. Push the DLPC solution back and forth 21 times, ending in the syringe that was empty to begin with. Note that some solution will be lost during the extrusion process.
FRAP solution before extrusion
FRAP solution after extrusion
FCS solution before extrusion
FCS solution after extrusion
After extrusion, aliquot the solution into Eppendorf tubes and store at -20ºC until ready to use
Bilayer formation
Bilayer formation
Equipment
Imaging Chamber
NAME
CoverWell
BRAND
635011
SKU
LINK
- Set hotplate to 30ºC
- Use double-sided kapton tape to adhere a substrate to the bottom of an imaging chamber
Substrates adhered to imaging chambers
- Place the substrate on the hotplate and deposit 30 μL of 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solution and 90 μL phosphate buffered saline onto the substrate
- Cover the substrate with a pipette box top or something else to protect from dust
- Let the substrate incubate for 10 minutes
TiO2-patterned substrates with DLPC solution and phosphate buffered saline
After 10 minutes of incubation, deposit 700 μL of phosphate buffered saline onto the substrate, flooding the imaging chamber
TiO2-patterned substrates after flooding the imaging chamber
- Wash the substrate by pipetting 500 μL off and then depositing it back on, moving around the substrate
- Repeat 15 times
Note
Take 500 μL of the solution off
Move to a different side of the substrate
Deposit the 500 μL of solution back into the imaging chamber
Move to a different side of the substrate and take 500 μL of solution off
Move to a different side of the substrate and deposit the 500 μL of solution back into the imaging chamber
After 15 washing steps, take 500 μL off and depose of it
Roll a coverslip over the imaging chamber
Note
For FCS measurements, coat the sides of the coverslip with clear nail polish to seal it to the imaging chamber
Substrates with coverslip
Image the samples to confirm bilayer formation. A representative image is shown below: black circles are TiO2 features deposited on the surface to inhibit bilayer formation (scale bar is 100um)