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EoE Sub Articles

1. Preparation of Large Unilamellar Vesicles (LUVs)
<ol>
	<li>Prepare LUVs for their use as cell membrane mimics for fluorescence leakage assay.</li>
	<li>Mix with a Hamilton glass syringe phosphatidylcholine (DOPC, 786.11 g/mol), sphingomyelin (SM, 760.22 g/mol) and Cholesterol (Chol, 386.65 g/mol) at the molar ratio 4:4:2. The lipid solution is obtained from a stock solution of each lipid solubilized in a methanol/chloroform (3/1; volume/volume) solvent at 25 mg/mL in a 25 mL glass round-bottom flask. Based on 4 &#181;mol of DOPC, 4 &#181;mol of SM, and 2 &#181;mol of Chol, the lipid solution is obtained from stock solution by mixing 126 &#181;L, 117 &#181;L, and 31 &#181;L, respectively. 
	CAUTION: Methanol is a toxic and inflammable solvent and chloroform is toxic and carcinogenic. Both should be handled with the appropriate protection under a hood.</li>
	<li>Evaporate methanol/chloroform using a rotary evaporator under vacuum during 45-60 min at 60 &#176;C until formation of a dried lipid film.</li>
	<li>Prepare two stock HEPES buffer solutions. Prepare HEPES buffer 1 by mixing 20 mM HEPES (238.3 g/mol) and 75 mM NaCl (58.44 g/mol) and adjust pH to 7.4. Prepare HEPES buffer 2 by mixing 20 mM HEPES and 145 mM NaCl and adjust pH to 7.4. HEPES buffers can be stored at 4 &#176;C for 1 month. 
	NOTE: It is recommended to check the osmolarity of the buffers using an osmometer.</li>
	<li>Prepare lipid hydration solution by dissolving membrane impermeable fluorescent dye-quencher couple, 8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt at 12.5 mM (ANTS, 427.33 g/mol) and p-xylene-bispyridinium bromide at 45 mM (DPX, 422.16 g/mol) in HEPES buffer solution. Mixing ANTS with DPX leads to a yellow-colored solution. To achieve the concentrations of 12.5 mM of ANTS and 45 mM of DPX, dissolve 21.4 mg and 76 mg, respectively in 4 mL of HEPES buffer 1. 
	NOTE: Lipid hydration solution can be stored for 2 weeks at 4 &#176;C by wrapping the tube with aluminum foil.</li>
	<li>Reconstitute multilamellar vesicles (MLV) by resuspending the dried lipid film with 1 mL of the lipid hydration solution and by vortexing until dissolution of the dried lipid film. Ensure that the solution is completely solubilized as small lipid aggregates will negatively impact the preceding steps. Also, check the wall of the glass round-bottom flask to ensure that there is no remaining lipid film. 
	NOTE: The solution will appear opalescent and light yellow after the solubilization.</li>
	<li>Subject the vesicles to five freeze/thaw cycles to obtain unilamellar vesicles. Perform each cycle by putting the glass round-bottom flask for 30 s in liquid nitrogen for freezing step, then leaving it in a water bath for 2 min for thawing step. 
	NOTE: The temperature of the bath water should be 5-10&#176;C higher than the melting temperature of the lipids.</li>
	<li>Prepare lipid extruder by inserting two filter supports preliminary humidified with HEPES buffer in each polytetrafluoroethylene (PTFE) extruder piece placed in the metal extruder canister.</li>
	<li>Put a HEPES humidified polycarbonate membrane (0.1 &#181;m pore size, 25 mm diameter) on the top of one filter support.</li>
	<li>Assemble the two metal extruder canisters and screw them.</li>
	<li>Place the assembled extruder in the holder and introduce a 1 mL syringe in the appropriate hole at the extremity of each polytetrafluoroethylene extruder piece. Extrusion corresponds to the passage of the liquid tested from one syringe to the other through the polycarbonate membrane.</li>
	<li>Test the extruder with 1 mL of HEPES buffer loaded in one of the 1 mL syringe to ensure that there are no leaks or problems.</li>
	<li>Replace the 1 mL HEPES buffer with the MLV sample.</li>
	<li>Perform extrusion by passaging the MLV sample from one syringe to the other through the polycarbonate membrane at least 21 times to obtain uniform LUVs of same size. 
	NOTE: Extrusion should be performed at a temperature higher than the melting temperature of the lipid mixture.</li>
</ol>
2. Purification of LUVs
<ol>
	<li>Prepare a column purification to remove non-encapsulated ANTS and DPX excess.</li>
	<li>Introduce cross-linked dextran gel (G-50) resuspended in aqueous medium with 0.01% NaN3 (65 g/mol) in a liquid chromatography column (Luer Lock, Non-jacketed, 1.0 cm x 20 cm, bed volume 16 mL) up to 1 cm below the top of the colorless part of the column.</li>
	<li>Open the tap and let the liquid flow to settle the cross-linked dextran gel.</li>
	<li>Wash the column by eluting with 20 mL of HEPES buffer 2 and discard the output flow of the column.</li>
	<li>Close the tap once the dead volume of solvent above the column is minimized (&#60;100 &#181;L) but sufficient to avoid any drying of the cross-linked dextran gel.</li>
	<li>Place the freshly extruded LUVs (yellow) on the column and let them enter into the cross-linked dextran gel.</li>
	<li>Continuously add HEPES buffer 2 to the column to perform the LUV purification.</li>
	<li>Elute approximately 2 mL of HEPES buffer 2 (do not forget to regularly fill the top of the column to avoid drying the cross-linked dextran gel): the free yellow ANTS and DPX solution migrates slower than the liposomes.</li>
	<li>Start collecting purified LUVs in tubes (1.5 mL).</li>
	<li>Observe the drops of eluent from the column and when they become opalescent, they contain liposomes. Change the tube to recover the LUV-containing fraction.</li>
	<li>Elute until the drops are no longer opalescent (~1 mL). Afterwards, elute another 0.5 mL in a separate fraction and then stop eluting. 
	NOTE: Standards are now available in a wide range of molecular weights, as kits or individual molecular weights to calibrate the elution volume of the LUVs.</li>
	<li>Wrap the tubes with the LUVs in aluminum foil to avoid bleaching of the fluorescence dye.</li>
	<li>Wash the column with 20 mL HEPES buffer 2.</li>
	<li>The LUVs can then be stored for a week at 4 &#176;C. 
	NOTE: As LUV stability might depend on LUV concentration and composition, as well as on ionic strength, the size of the LUVs should be controlled using a dynamic light scattering (DLS) instrument (see section 4. Characterization of LUV Size and Homogeneity) before each test.</li>
</ol>
3. Quantifying the concentration of LUVs
<ol>
	<li>Estimate LUV concentration by a phospholipid quantification kit, which enables the evaluation of choline concentration. This assay might be applied when phospholipids with choline containing polar head is substantial (&#62;50% of the LUVs).</li>
	<li>Prepare the color reagent by dissolving 18 mg of chromogen substrate in 3 mL of buffer provided.</li>
	<li>Load a polystyrene cuvette, 10 x 10 x 45 mm, with 3 mL of color reagent.</li>
	<li>Use the pure color reagent as blank condition (Blank). Add 20 &#181;L of LUV sample (Test) or 20 &#181;L of standard solution of known choline concentration (Standard).</li>
	<li>Mix well and incubate for 5 min at 37 &#176;C all conditions (Blank, Test, and Standard).</li>
	<li>Measure the absorbance (optical density, OD) of the test sample and standard solution with the blank solution as the control at 600 nm with a spectrophotometer.</li>
	<li>Check the OD values which enable to estimate the lipid concentration of the LUVs, C[LUV], in choline equivalent compared to the standard of known concentration.</li>
	<li>Perform the calculation using the following equation: 
	C[LUV] (mol / l) = (OD Sample / OD Standard) x C[Standard] (mol / l) 
	NOTE: The phospholipid quantification kit provided a Choline Chloride (139.6 mg/l) standard solution at 54 mg/dL corresponding to molar concentration of C[Standard] = 3.87 mmol/L. OD Sample and OD Standard are the absorbances measured at 600 nm for the LUV and Choline solutions, respectively.</li>
</ol>
4. Characterization of LUV size and homogeneity
<ol>
	<li>Perform a measurement using a DLS instrument in order to determine the LUV size (in nm) and polydispersity index (PdI).</li>
	<li>Program the appropriated &#34;standard operation procedure&#34; (SOP) by indicating the viscosity of the solvent/buffer and the used cuvette.</li>
	<li>Place 500 &#181;L of the LUV solution in a polystyrene semi-micro cuvette.</li>
	<li>Insert the polystyrene semi-micro cuvette in a DLS instrument.</li>
	<li>At room temperature, measure the size distribution in terms of mean size (Z-average) of the particle distribution and of homogeneity (polydispersity index, PdI).</li>
	<li>All the results are obtained from two independent measurements performed each in three repetitive cycles. 
	NOTE: Standard values for LUVs will be a mean size of 137 &#177; 7 nm with a PdI of 0.149 &#177; 0.041.</li>
</ol>
5. Preparing peptide solutions
<ol>
	<li>Prepare a stock solution of the peptide, which should be analyzed for the leakage assay.</li>
	<li>Dissolve peptide powder (&#62;95% purity) in pure water (e.g., 1 mg peptide in 500 &#181;L pure water). 
	NOTE: It is recommended to dilute peptides in pure water and to avoid dimethyl sulfoxide (DMSO) solubilization, which could induce artifacts (e.g., membrane permeabilization).</li>
	<li>Vortex the peptide solution for 5 s.</li>
	<li>Sonicate the peptide solution in a water sonication bath for 5 min and then centrifuge for 5 min at 12,225 x g. Collect the supernatant for concentration determination.</li>
	<li>Measure the absorbance at 280 nm of three independent peptide dilutions and then calculate peptide concentration using its molar extinction coefficient &#949; (depending on tryptophan and tyrosine content in the peptide sequence) and Beer-Lambert rule. 
	NOTE: If the peptide contains tryptophan and tyrosine, the molar extinction coefficient &#949; is computed on the basis of Tryptophan &#949; = 5,690 M-1cm-1 and Tyrosine &#949; = 1,280 M-1cm-1. If the peptide sequence contains no tryptophan or tyrosine, other colorimetric assay could be performed to measure the concentration (e.g., BCA or Bradford).</li>
	<li>Dilute the peptide solution in pure water to a final solution of 100 &#181;M and store at 4 &#176;C. 
	NOTE: In pure water, no peptide degradation occurs during the 4 &#176;C storage. However, peptide concentration should be measured every 2 weeks to ensure that no water evaporation occurs.</li>
</ol>
6. Fluorescence leakage assay
<ol>
	<li>Fluorescence leakage assay is measured on a spectrofluorometer at room temperature. Excitation and emission wavelength are fixed at Ex = 360 nm &#177; 3 nm and Em = 530 nm &#177; 5 nm, respectively.</li>
	<li>Dilute LUVs in 1 mL HEPES buffer 2 to a final concentration of 100 &#181;M in a quartz fluorescence cuvette. Add a magnetic stirrer to homogenize the solution during experiment.</li>
	<li>Measure the LUVs alone during the first 100 s, between t = 0 s and t = 99 s in order to access the background fluorescence. 
	NOTE: LUVs alone could also be measured during the whole experiment (15 min) in order to access background fluorescence and potential leaks.</li>
	<li>Thereafter, measure leakage as an increase in fluorescence intensity upon addition of aliquots of peptide solution for the next 900 s (15 min). This protocol is carried out for each concentration of peptide tested from 0.1 &#181;M to 2.5 &#181;M.</li>
	<li>Finally, 100% fluorescence was achieved by solubilizing the LUVs by addition of 1 &#181;L of Triton X-100 (0.1%, v/v), resulting in the completely unquenched probe between t = 1,000 s and t = 1,100 s.</li>
</ol>
7. Quantification of the leakage
<ol>
	<li>Suppress values obtained after t = 1,090 s in order to keep the same number of points for each tested condition.</li>
	<li>Calculate the minimal fluorescence, Fmin, by making the average of 50 points between t = 0 s and t = 49 s (LUVs alone).</li>
	<li>Calculate the maximal fluorescence, Fmax, by making the average of 50 points between t = 1,041 s and t = 1,090 s (LUVs with Triton X-100).</li>
	<li>Calculate the leakage percentage (%Leak) at each time point (t = x), according to the following equation: 
	%Leak(t=x) = (F(t=x) - Fmin) / (Fmax - Fmin) x 100</li>
	<li>Calculate the average and standard deviation for values obtained with different LUV preparation (n &#8805; 2) for the same condition.</li>
	<li>Plot the leakage percentage, %Leak(t=x), in function of time (s).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25 mL glass round-bottom flask</td>
			<td>Pyrex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS)</td>
			<td>Invitrogen</td>
			<td>A350</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>288306</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC (dioleoylphosphatidylcholine)</td>
			<td>Avanti Polar</td>
			<td>850375P</td>
			<td>Protect from air</td>
		</tr>
		<tr>
			<td>Extruder</td>
			<td>Avanti Polar</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorimeter</td>
			<td>PTI Serlabo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;L glass syringe</td>
			<td>Hamilton</td>
			<td>705N</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>LabAssay Phospholipid</td>
			<td>WAKO</td>
			<td>296-63801</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid chromatography column</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carlo Erba</td>
			<td>414902</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclepore polycarbonate membrane (0.1 &#181;m pore size, 25 mm diameter)</td>
			<td>Whatman</td>
			<td>800309</td>
			<td></td>
		</tr>
		<tr>
			<td>polystyrene cuvette, 10 x 10 x 45 mm</td>
			<td>Grener Bio-One</td>
			<td>614101</td>
			<td></td>
		</tr>
		<tr>
			<td>polystyrene semi-micro cuvette, DLS</td>
			<td>Fisher Scientific</td>
			<td>FB55924</td>
			<td></td>
		</tr>
		<tr>
			<td>p-xylene-bispyridinium bromide (DPX)</td>
			<td>Invitrogen</td>
			<td>X1525</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>quartz fluorescence cuvette</td>
			<td>Hellma</td>
			<td>109.004F-QS</td>
			<td></td>
		</tr>
		<tr>
			<td>rotavapor system</td>
			<td>Heidolph</td>
			<td>Z334898</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G-50 resin</td>
			<td>Amersham</td>
			<td>17-0042-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chlorid (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator bath USC300T</td>
			<td>VWR</td>
			<td>142-6001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sphingomyelin</td>
			<td>Avanti Polar</td>
			<td>860062P</td>
			<td>Protect from air</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Eromedex</td>
			<td>2000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetaziser NanoZS</td>
			<td>Malvern</td>
			<td>ZEN3500</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence leakage assay to study cell-penetrating peptide interaction with membrane

Source: Konate, K. et al. <a href="https://app.jove.com/v/62028">Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide.</a> J. Vis. Exp. (2020)
This video discusses the fluorescence leakage assay to study the interactions of cell-penetrating peptides with cell membranes using cell membrane mimics &#8212; large unilamellar vesicles, or LUVs, encapsulated with a fluorescent dye and a quencher.

Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane

Source: Konate, K. et al. Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide. J. Vis. Exp. (2020)
This video ...

Cell-penetrating peptides, CPPs, short, positively charged peptides, can interact with and cross cellular membranes, facilitating the delivery of cell-impermeable cargoes.&#160;
To study the membrane permeabilization potential of a test CPP in vitro using a cell membrane mimic, begin with a large unilamellar vesicle, LUV, suspension.
LUVs comprise a phospholipid bilayer loaded with a membrane-impermeable fluorescent probe and corresponding quencher pair. Within the vesicles, the quencher interacts with the fluorescent probe due to their close proximity, causing fluorescence quenching.&#160;
Pipette the LUV suspension into a quartz fluorescence cuvette, and dilute using buffer. Add a magnetic bar. Transfer to a spectrofluorometer with magnetic stirring to prevent LUV sedimentation during the run. Determine the background fluorescence to eliminate any LUV leaks.
Add the desired concentration of CPP to the LUV suspension at regular intervals.
Based on the hydrophobicity and charge of the CPPs&#39; amino acid residues, they perturb the LUV lipid membrane, leading to membrane penetration and permeabilization.
The vesicle membrane damage causes leakage of the fluorescent probe and quencher into the surrounding solution, increasing their spatial separation. As a result, the probe exhibits fluorescence.
Further CPP solution addition leads to increased LUV permeabilization and fluorescence intensity. An increase in fluorescence intensity with CPP addition suggests the peptide&#39;s membrane permeabilizing potential.

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Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

216 Views. Source: Konate, K. et al. Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide. J. Vis. Exp. (2020) This video discusses the fluorescence leakage assay to study the interactions of cell-penetrating peptides with cell membranes using cell membrane mimics — large unilamellar vesicles, or LUVs, encapsulated with a fluorescent dye and a quencher. 

Video: Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane - Concept

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

JoVE Journal

Biochemistry

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Bioengineering

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane

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