Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results

Boban, Zvonimir; Mardešić, Ivan; Subczynski, Witold K.; Raguž, Marija

doi:10.3390/membranes11110860

Cited by 18 publications

(18 citation statements)

References 110 publications

(301 reference statements)

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“…Compared to vesicles grown using the gentle hydration method, the electroformation method reduces the compositional heterogeneity of the vesicles [ 6 ] and increases the proportion of unilamellar vesicles [ 3 ]. The method has evolved significantly over the years, with many potential pitfalls identified and protocol modifications tested [ 7 , 8 , 9 ]. The most important issues are related to the use of organic solvents during lipid film deposition, reproducibility of the conventional film deposition technique, and the step in which the lipid film is completely dried.…”

Section: Introductionmentioning

confidence: 99%

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion

et al. 2023

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Giant unilamellar vesicles (GUVs) are artificial membrane models which are of special interest to researchers because of their similarity in size to eukaryotic cells. The most commonly used method for GUVs production is electroformation. However, the traditional electroformation protocol involves a step in which the organic solvent is completely evaporated, leaving behind a dry lipid film. This leads to artifactual demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the formation of the lipid bilayer, resulting in a decrease of Chol concentration in the bilayer compared to the initial lipid solution. We propose a novel electroformation protocol which addresses this issue by combining the rapid solvent exchange, plasma cleaning and spin-coating techniques to produce GUVs from damp lipid films in a fast and reproducible manner. We have tested the protocol efficiency using 1/1 phosphatidylcholine/Chol and 1/1/1 phosphatidylcholine/sphingomyelin/Chol lipid mixtures and managed to produce a GUV population of an average diameter around 40 µm, with many GUVs being larger than 100 µm. Additionally, compared to protocols that include the dry film step, the sizes and quality of vesicles determined from fluorescence microscopy images were similar or better, confirming the benefits of our protocol in that regard as well.

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Section: Introductionmentioning

confidence: 99%

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion

et al. 2023

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“…The dried film is then hydrated, and an alternating current applied to detach the lipid film from the surface and form GUVs. Although quite simple at first glance, the method is influenced by many parameters, requiring optimization depending on the experimental setup [ 2 ]. We focus on the effect of lipid composition, lipid film thickness, and electrical parameters (frequency and voltage) using a ternary mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine/sphingomyelin/cholesterol (POPC/SM/Chol) ( Figure 1 ).…”

Section: Introductionmentioning

confidence: 99%

Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures

et al. 2022

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Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency–voltage combinations in the range of 2–6 V and 10–100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

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“…First, we investigated if our methods could be used to track GUV size and number during swelling-based GUV formation. GUV swelling methods include electroformation and gel-assisted swelling and are by far the most popular formation methods, as they are fast and easy and yield large numbers of GUVs. ,,, In these experiments, lipids are first dried on a surface, which can be a hydrogel, an electrode, glass, Teflon, or a porous material. Subsequent addition of a swelling solution leads to swelling of the lipid film and formation of large numbers of GUVs that are closely packed above the swelling surface (Figure A, top).…”

Section: Resultsmentioning

confidence: 99%

“…Despite the experimental ease of producing and imaging GUVs, their quantitative image analysis has received relatively little attention . Typically, GUVs are either manually detected in the image and afterward (manually) processed to extract data, ,− or custom-made scripts are used to process specific data sets and generate a predefined set of output parameters. ,,− While general image analysis software (e.g., ImageJ) can be used with GUV microscopy data, there are no dedicated modules or plugins for GUV detection and analysis, requiring for the user to adapt the built-in methods and, in case of automated analysis, programming knowledge is often needed.…”

Section: Introductionmentioning

confidence: 99%

DisGUVery: A Versatile Open-Source Software for High-Throughput Image Analysis of Giant Unilamellar Vesicles

2022

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Giant unilamellar vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVery’s three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple predefined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape, and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of nonspherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in synthetic cell research.

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Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results

Cited by 18 publications

References 110 publications

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion

Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures

DisGUVery: A Versatile Open-Source Software for High-Throughput Image Analysis of Giant Unilamellar Vesicles

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