Name: Cell Electrofusion Visualized with Fluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2010-07-01T17:00:00.000+00:00
Duration: 5 min 2 s
Description: Learn cell electrofusion using a modified adherence method with mouse melanoma cells and fluorescence microscopy. This protocol enables efficient hybrid cell creation and quantitative fusion yield assessment for biological research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the Slovenian Research Agency (project J2-9764 and programme P2-0249). This video represents supplementary material for the "Electroporation-based Technologies and Treatments" scientific workshop and postgraduate course, organized by the Faculty of Electrical Engineering at the University of Ljubljana, Slovenia.

I. Loading the cells with Cell trackers CMFDA and CMRA
<ol>
 <li>Experiments were performed on previously prepared cells of mouse melanoma cell line (B16-F1). Cells are grown in two separated 25 cm2 culture flasks (TPP, ZDA) to 70-80 % confluence in DMEM culture medium (Dulbecco's modified Eagle's medium) supplemented with 10 % fetal bovine serum, 0.15 mg/ml L-glutamine, 16 mg/ml gentamicin (all from Sigma-Aldrich, Germany), 200 units/ml crystacillin (Pliva, Croatia), and incubated in 5% CO2 at 37°C.</li>
 <li>Prepare two 10 mM stock solutions of Cell trackers (Invitrogen, USA) by adding 10.76 μl and 9 μl (for Green CMFDA and for Orange CMRA, respectively) of DMSO (Sigma-Aldrich, Germany) to 50 μg of the dye in the original Invitrogen vial. The stock solution can be stored in a refrigerator at 4°C for few months. Before starting the experiments, warm up the solution until the crystals of DMSO dissolve.</li>
 <li>Prepare bicarbonate-free Krebs-Hepes buffer (130 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4 , 1.2 mM KH2PO4, 11.7 mM D-glucose, 1.3 mM CaCl2, 10 mM HEPES, pH 7.4). In two 15 ml Eppendorf tubes separately mix 2.1 μl of each stock solution (10 mM CMFDA or CMRA, respectively) in 3 ml of bicarbonate-free Krebs-Hepes buffer. This yields a "loading solution" containing approximately 7 μM CMFDA (or CMRA).</li>
 <li>Rinse cells twice with bicarbonate-free Krebs-Hepes buffer and then insert loading solutions into the flasks. Incubate cells for 30 minutes in 5% CO2 at 37 °C. During this first incubation reagents pass freely through cell membranes, but once inside the cell, the reagents are transformed into cell-impermeant fluorescent reaction products.</li>
 <li>After first incubation, rinse and incubate cells with culture medium for another two hours in 5% CO2 at 37°C.</li>
 <li>Trypsinize cells in both flasks (loaded with CMFDA and CMRA) and mix red and green cells together in a ratio 1:1 in a 50 ml centrifuge tube (TPP, ZDA). Adjust cell concentration to 5 x 106 cells/ml by dilution with DMEM medium or by concentration with centrifuge. Place a 20 μl drop of cell suspension in each well of 24 multiwell plate (TPP, ZDA). Incubate cells in 5 % CO2 at 37 °C for 20 min to allow them to slightly attach to the surface of the well and establish cell contacts.</li>
</ol>
II. Electrofusion
<ol>
 <li>Prepare isoosomolar potassium phosphate buffer (10 mM KH2PO4, 10 mM K2HPO4, 1mM MgCl2, 250 mM sucrose) and hypoosmolar potassium phosphate buffer (10 mM KH2PO4, 10 mM K2HPO4, 1mM MgCl2, 75 mM sucrose).</li>
 <li>Place the multiwell with cells onto the microscope stage, position the electrodes at the bottom of the well and connect them to the pulse generator.</li>
 <li>Wash the cells with 1 ml of isoosomolar potassium phosphate buffer. Add 350 μl of hypoosmolar potassium phosphate buffer in order to induce cell swelling. The buffer should cover the electrodes.</li>
 <li>Leave cells in hypoosmolar buffer for 2 minutes before applying electric pulses. During this time influx of water molecules into the cells due to osmotic imbalance between the interior and the exterior of the cells cause an increase of cell volume. Electric pulses should be applied when cells are close to their maximum volumes, before regulatory volume decrease start.</li>
 <li>To achieve optimal electrofusion and maintain cell viability, optimal parameters of electric pulses should be used. These depend on cell line used [1]. In this experiment a train of 8 rectangular pulses (each with duration of 100 μs at 1 Hz) is applied to each sample, using electroporation device (in our case Cliniporator, IGEA, Italy). The pulses are delivered to two parallel Pt/Ir wire electrodes with 0.8 mm diameter and 5 mm distance between them, creating an electric field of approximately 1200 V/cm between the electrodes in each well, except for the control well.</li>
 <li>Leave the cells undisturbed for 10 minutes after pulse delivery. Determine the fusion yield by means of fluorescent and phase contrast microscopy.</li>
</ol>
III. Image acquisition and determination of the fusion yield
<ol>
 <li>The cells are observed using a fluorescence microscope (in our case Zeiss AxioVert 200, Zeiss, Germany) equipped with an objective (x20) and a cooled CCD camera (VisiCam 1280, Visitron, Germany). The images are acquired in MetaMorph 7.1.1 (Molecular Devices, USA), but other similar acquisition software can also be used. CMFDA is excited with a monochromator (Polychrome IV, Visitron, Germany) at 492 nm and CMRA at 548 nm. The fluorescence of CMFDA and CMRA is acquired using two emission filters, one centered at 535 nm (HQ535/30m, for CMFDA) and the other centered at 510 nm (D605/55m, for CMRA, both Chroma, USA). The use of dichroic mirror (Q515LP) prevented the channel cross talk.</li>
 <li>Acquire three images (phase contrast, red and green fluorescence) for five randomly chosen fields in each well. Create three channel images from each image triplet. In such image fluorescing cytoplasm can be seen together with the cell membranes. Fused cells can thus be easily determined [Figure 1].</li>
 <li>Count all three types of cells (red, green and dually fluorescent) in each three channel image. Determine the percentage of dually fluorescent cells by dividing the number of dually fluorescent cells with the number of all cells in each image. Fusion yield is defined as the percentage of dually fluorescent cells multiplied by 2 since half of fused cells are not detected (when cells of the same color fuse). </li>
</ol>
Representative Results
<img src="/files/ftp_upload/1991/1991fig1.jpg" alt="Yeast cell microscopy image; fluorescence highlights red and green proteins within cells." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Three channel microscopy image of B16F1 cells after electrofusion: phase contrast, CMRA fluorescence (excitation at 548 nm) and CMFDA fluorescence (excitation at 492 nm), objective magnification 20x

<ol><li>Čemazar, M., Jarm, T., Miklavčič, D., Maček-Lebar, A., Ihan, A., Kopitar, N. A., Serša, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+electric-field+intensity+on+electropermeabilization+and+electrosensitivity+of+various+tumor-cell+lines+in+vitro%5BTitle%5D)+AND+%22Electro.+Magnetobiol%22%5BJournal%5D)">Effect of electric-field intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro.</a> Electro. Magnetobiol. 17, 263-272 (1998).</li>
<li>Trontelj, K., Reberšek, M., Kandušer, M., čurin šerbec, V., Miklavčič, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+bulk+cell+electrofusion+in+vitro+for+production+of+human-mouse+heterohybridoma+cells%5BTitle%5D)+AND+%22Bioelectrochemistry%22%5BJournal%5D)">Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells.</a> Bioelectrochemistry. 74, 124-129 (2008).</li>
<li>Rols, M. -P., Teissié, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+electrically+induced+permeabilization+and+fusion+of+Chinese+hamster+ovary+cells+by+osmotic+pressure%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure.</a> Biochemistry. 29, 4561-4567 (1990).</li>
<li>Neil, G., Zimmermann, U. Electrofusion. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNeil%2C+G+%22Methods+in+Enzymology%22">Methods in Enzymology</a>. 220, 174-196 Forthcoming.</li>
<li>Abidor, I. G., Li, L. -H., Hui, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+of+cell+pellets%3A+II.+Osmotic+properties%2C+electroporation%2C+and+related+phenomena%3A+Membrane+interactions%5BTitle%5D)+AND+%22Biophysical+journal%22%5BJournal%5D)">Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: Membrane interactions.</a> Biophysical journal. 67, 427-435 (1994).</li>
<li>Jaroszeski, M. J., Gilbert, R., Fallon, P. G., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanically+facilitated+cell-cell+electrofusion%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Mechanically facilitated cell-cell electrofusion.</a> Biophys J. 67 (4), 1574-1581 (1994).</li>
<li>Ramos, C., Bonefant, D., Teissié, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+hybridization+by+electrofusion+on+filters%5BTitle%5D)+AND+%22Analytical+biochemistry%22%5BJournal%5D)">Cell hybridization by electrofusion on filters.</a> Analytical biochemistry. 302, 213-219 (2002).</li>
<li>Cao, Y., Yang, J., Yin, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+high-throughput+cell+electrofusion+in+a+microelectrode-array+chip%5BTitle%5D)+AND+%22Microfluid+Nanofluid%22%5BJournal%5D)">Study of high-throughput cell electrofusion in a microelectrode-array chip.</a> Microfluid Nanofluid. 5, 669-675 (2008).</li>
<li>Ušaj, M., Trontelj, M., Kandušer, M., Miklavčič, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+size+dynamics+and+viability+of+cells+exposed+to+hypotonic+treatment+and+electroporation+for+electrofusion+optimization%5BTitle%5D)+AND+%22Radiol.+Oncol%22%5BJournal%5D)">Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization.</a> Radiol. Oncol. 43, 108-119 (2009).</li>
<li>Teissié, J., Ramos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlation+between+electric+field+pulse+induced+long-lived+permeabilization+and+fusogenicity+in+cell+membranes%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.</a> Biophys. J. 74, 1889-1898 (1998).</li>
</ol>

No conflicts of interest declared.

The ability of cell membranes to fuse non-specifically, e.g., by external electric fields, is important for biotechnology, medicine and research in biology. Such nonspecific fusion enables production of highly valuable hybrid cells and their products, such as monoclonal antibodies, and provides information about fundamental mechanisms of fusion [2]. Electrofusion is a potentially very effective method since it can be properly adjusted to different types of cells. Electrofusion is achieved when cells in close physical contact are brought into their fusogenic state (prone to fusion) by means of high-voltage electric pulses. The efficiency of electrofusion depends on various parameters that affect two parts of the electrofusion process. First part of the electrofusion process is achievement of the close physical contact between cells, which can be obtained with different methods [3-8]. Adherence method (growing cells to confluence) can be used efficiently due to spontaneously established cell contacts in large zones between cells; however, it produces very large fused cells with many nuclei. We are using the modified adherence method, where smaller cells (with 2 to 5 nuclei), which are more likely to survive and proliferate, are obtained (Figure 1). Contact between cells also benefit from osmotic swelling of cells, due to osmotic treatment used in the experiment [9]. Second part of the electrofusion process is the achievement of the fusogenic state of the cell membranes. Fusogenic state correlates well with electropermeabilized state of the membrane (cells are non-specifically permeabilized to molecules that normally cannot pass through intact membrane) and is governed by the same parameters of the electric pulses (amplitude, length, number and frequency) [10]. The values of electrical parameters needed for optimal electroporation [1] and electrofusion differ between different cells and depend on cells size and their biological properties. Electrical parameters thus need to be optimized for different cell lines, which are used as fusion partners, to obtain fusion.

<table><tbody><tr><td>CMRA</td><td>Reagent</td><td>Invitrogen</td><td>C34551</td><td>Cytosolic fluorescent dye</td></tr><tr><td>CMFDA</td><td>Reagent</td><td>Invitrogen</td><td>C7025</td><td>Cytosolic fluorescent dye</td></tr><tr><td>DMSO</td><td>Reagent</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DMEM </td><td>Reagent</td><td>Sigma-Aldrich</td><td>D5671</td><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium</td></tr><tr><td>Fetal calf serum</td><td>Reagent</td><td>Sigma-Aldrich</td><td>F4135</td><td></td></tr><tr><td>L-glutamine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>crystacillin</td><td>Reagent</td><td>Pliva</td><td>625110</td><td>antibiotic</td></tr><tr><td>gentamicin</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G1397</td><td>antibiotic</td></tr><tr><td>Hepes</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Reagent</td><td>Merck &amp;amp; Co., Inc.</td><td>A124873 927</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Reagent</td><td>Sigma-Aldrich</td><td>4248</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M-8266</td><td></td></tr><tr><td>NaCl</td><td>Reagent</td><td>Fluka</td><td>71382</td><td></td></tr><tr><td>KCl</td><td>Reagent</td><td>Merck &amp;amp; Co., Inc.</td><td>A154336 908</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M2643</td><td></td></tr><tr><td>D-glucose</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Reagent</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>sucrose</td><td>Reagent</td><td>Sigma-Aldrich</td><td>16104</td><td></td></tr><tr><td>Electric pulse generator</td><td>Tool</td><td>IGEA</td><td></td><td>Cliniporator VITAE</td></tr><tr><td>Multiwell plate</td><td>Tool</td><td>Techno Plastic Products</td><td>92424</td><td></td></tr><tr><td>50 ml centrifuge tube</td><td>Tool</td><td>Techno Plastic Products</td><td>91050</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>Tool</td><td>Techno Plastic Products</td><td>91015</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; culture flask</td><td>Tool</td><td>Techno Plastic Products</td><td>90026</td><td></td></tr><tr><td>Electrodes</td><td>Tool</td><td>Custom Made</td><td></td><td>Pt/Ir</td></tr></tbody></table>

cell electrofusion visualized with fluorescence microscopy

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Electro fusion involves application of short high voltage electric pulses to cells in close context. In this video, we demonstrate electro fusion of cells in vitro by means of modified Adherence.Method. Experiments were performed on mouse Melanoma cells, BF one.For the experiment, follow these steps. Grow cells in two separate culture flasks to 80%confluence. Separately, add 2.1 microliters of each stock solution.10 millimolar C-M-F-D-A or CMRA respectively to three milliliters of Krebs Hess Buffer in a centrifuge tube. Rinse cells twice with Krebs Hess buffer and then insert loading solutions into the flasks. Loading solutions contain approximately seven Micromolar C-M-F-D-A or CMRA respectively.Incubate cells for 30 minutes in a controlled atmosphere. During this first incubation, regions pass freely through cell membranes into the cytosol where they are transformed into membrane IMP permeant reaction products. After this first incubation, rinse and then incubate the cells with culture medium for another two hours.The cells are observed using a fluorescence microscope equipped with a cooled CCD camera in the acquisition software. Set the excitation wavelength to 548 nanometers and choose an appropriate band pass filter threat fluorescence image of the cells loaded with CMRA for the cells loaded with C-M-F-D-A. Set the excitation wavelength to 492 nanometers and choose a band pass filter for green fluorescence image trypsin eyes, and count the cells in both flasks and mix red and green cells together In a ratio one to one, adjust cell concentration to 5 million cells per milliliter.Place a 20 microliters drop of cell suspension in the middle of each well. In a 24 multi-well plate incubate cells in a controlled atmosphere for 20 minutes to allow cells to slightly attach to the surface of the well in a monolayer and establish cell contacts between themselves. Place the multi-well with cells onto the microscope stage.Position the electrodes at the bottom of the well and connect them to the pulse generator To achieve optimal electro fusion and maintain cell viability, adequate parameters of electric pulses should be used. These parameters depend on cell line and should be determined in preliminary experiments. Remove the culture medium and wash the cells with one milliliter ISO omu potassium phosphate buffer at 350 microliters hypos Miller potassium phosphate buffer in order to induce cell swelling.Leave cells in hyperosmal buffer for two minutes before applying electric pulses. Electric pulses should be applied when cells are close to their maximum volumes. That is before they start regulatory volume.Decrease in our experiment. A train of eight rectangular pulses with duration of 100 microseconds at one hertz was applied. After pulse Delivery.Leave the cells undisturbed for 10 minutes after 10 minutes. Determined Diffusion yield by means of face contrast and fluorescent microscope. E acquire three images, face contrast, red and green fluorescence at five randomly chosen fields of view.In each, well create three channel images from each image triplet in image J software in order to improve visual quality of images to pre-processing steps can be applied to the original images, background, subtraction, and contrast enhancements with default parameter sets. Finally, three channel images are composed using RGB to gray image J plugin. In such image, the resting cytoplasm can be seen together with cell membranes.Thus few cells can easily be determined in the three channel image count all three types of cells, red, green, and dually fluorescent. Determine the percentage of dually fluorescent cells by dividing the number of dually fluorescent cells with a number of all cells in each image. Fusion yield is then defined as the percentage of dually fluorescent cells multiplied by two.Since half of the view cells are not detected when cells of the same color views.

cell-electrofusion-visualized-with-fluorescence-microscopy

Research

JoVE Journal

Biology

We described structural features of the Glia-neuromuscular synapses in a novel  Inside-out  tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Source: Aigrain, L., et al. Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation. J. Vis. Exp. (2015)The video demonstrates the delivery of fluorescent biomolecules into electrocompetent microbial cells using electroporation. The process begins with the introduction of fluorophore-tagged DNA or proteins into a chilled cell suspension. A high-voltage pulse is then applied to create temporary membrane pores, enabling biomolecule entry into the...

Electroporation-Based Delivery of Fluorescent Biomolecules into Bacterial Cells

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals.  Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA Electroporation, Isolation and Imaging of Myofibers

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on Förster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion.

Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Learn cell electrofusion using a modified adherence method with mouse melanoma cells and fluorescence microscopy. This protocol enables efficient hybrid cell creation and quantitative fusion yield assessment for biological research.

Cell Electrofusion Visualized with Fluorescence Microscopy

Explore More Videos