This work was supported by University of Alberta Faculty of Medicine and Dentistry, The Friends of Garrett Cumming Research Chair Fund, HM Toupin Neurological Science Research Chair Fund, Muscular Dystrophy Canada, Canada Foundation for Innovation (CFI), Alberta Advanced Education and Technology (AET), Canadian Institutes of Health Research (CIHR), Jesse's Journey - The Foundation for Gene and Cell Therapy, the Women and Children's Health Research Institute (WCHRI), and Alberta Innovates Health Solutions (AIHS).
We would like to thank Dr. Steven Laval for supplying us with the full-length dysferlin plasmid. We would also like to thank Dr. Katsuya Miyake for technical advice.

Human fibroblast cells used in this study were used with approval from the Human Ethics Committee of the Faculty of Medicine and Dentistry at the University of Alberta.
1. Transfection of cells with full-length DYSF plasmid
<ol>
	<li>Culture fibroblast cells in a T225 flask containing 40 mL of growth media - 36 mL Dulbecco&#39;s Modified Eagle Medium (DMEM), 4 mL of fetal bovine serum (FBS), and 20 &#181;L of penicillin/streptomycin (P/S) - in a CO2 incubator at 37&#176;C. 
	...

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The authors have nothing to disclose.

Two-photon laser wounding of the cell membrane is a precise and versatile technique for assessing the dynamics of membrane resealing in vitro. In this article, we described a protocol for determining cell membrane resealing ability in dysferlinopathy patient cells using the two-photon laser wounding assay. Our results show that dysferlinopathy patient cells are defective in cell membrane resealing, which is consistent with findings by other researchers and which further highlights the striking similarities in ce...

The cell membrane of eukaryotic cells consists of a double-layer of protein-studded phospholipids which defines the intra/extracellular environment of the cell and is essential for maintaining cellular homeostasis and cell survival. Cell membrane injuries arising from mechanical or chemical insults are commonplace in various mammalian cell types, including skeletal and cardiac muscle, stomach, and lung cells1,2,3,4. In addition to injuries consequent to daily physiological function, cell membranes can also be damaged by environmental insults, bacterial toxins, and ischemic reperfusion5. Failure to reseal ruptures in the cell membrane leads to an unregulated influx of extracellular Ca2+- along with other potentially toxic extracellular components - into the cell, triggering downstream signal cascades which can quickly result in cell death1,4,5,6.
To date, there have been several proposed models for membrane repair in cells. Different repair mechanisms may be activated depending on the size and nature of the membrane rupture. For example, it is suggested that cell membranes may utilize lateral flow or protein clogging to repair small disruptions (&#60;1 nm). The lateral fusion model proposes that membrane ruptures are rapidly mended through lateral recruitment of dysferlin-containing membrane7, while the protein clogging model suggests that small perforations are mended through protein aggregations (mostly annexins)8. Conversely, larger membrane lesions trigger Ca2+-dependent, dysferlin-mediated vesicle fusion and formation of a repair patch. In the repair patch model, a rapid Ca2+ influx into the cell triggers the recruitment of multiple proteins (dysferlin, annexins, mitsugumin-53, and EDH proteins) which form a complex or &#34;patch&#34;, along with a fusion of intracellular vesicles or lysosomes at the site of membrane damage4,9,10,11,12. It is important to note that these models are not necessarily mutually exclusive and may work in concert to facilitate membrane repair. Failure to properly reseal cell membrane damage is associated with multiple disease states, including muscular dystrophy (dysferlinopathy - including Miyoshi myopathy, limb-girdle muscular dystrophy type IIB, and distal myopathy with anterior tibial onset)13, cardiomyopathy14, and Chediak-Higashi syndrome (CHS)15.
Given that proper cell membrane integrity and resealing plays such an important role in health and disease, a deeper understanding of the underlying molecular mechanisms of cell membrane repair would be beneficial in the search for novel therapeutic strategies. It is, therefore, necessary to possess appropriate experimental techniques to&#160;monitor the kinetics and evaluate the ability of cell membrane repair. Several in vitro methods for modeling membrane repair have been designed. One strategy involves mechanical injury, which can be facilitated through cell scraping with a pipette/surgical blade/razor or by rolling glass beads over the cells16. However, this type of mechanical injury generates larger lesions, and creates a high degree of variation in cell injury, both within and between cultures.
Another method of generating membrane wounds is laser ablation by two-photon microscopy. In contrast to traditional laser confocal microscopy that employs single photon excitation, the two-photon laser simultaneously uses two long-wavelength, low-energy photons to facilitate the excitation of a high-energy electron17. This non-linear process results in excitation exclusively within the focal plane and not along the entire light path17 (Figure 1). This reduced excitation volume helps to minimize photodamage when imaging live cells18,19. Researchers are therefore able to generate precise lesions in the cell membrane and monitor membrane resealing in real time by using fluorescent dyes and observing changes in the fluorescence intensity as the cell membrane ruptures and then reseals.
This approach has been used repeatedly to study membrane wounding in vitro and in vivo and in multiple cell types20,21. For example, membrane repair defects in fibroblasts and myotubes derived from dysferlinopathy patients were assessed using this technique22,23. Also, single muscle fibers isolated from mice were used to monitor repair patch formation13,24,25. The movement of fluorescently tagged proteins can also be observed during membrane repair in single muscle fibers9. Furthermore, the process of sarcolemmal repair following two-photon laser wounding in zebrafish embryos can be observed in real-time in vivo26.
In this article, we outline a methodology for assessing cell membrane repair dynamics in fibroblasts using two-photon laser wounding, although this methodology can be applied to various cell types for the purpose of quantifying plasma membrane resealing ability in vitro. In this method, cells are incubated with FM4-64, a lipophilic, cell-impermeable dye which quickly fluoresces as it binds to negatively charged phospholipids within the cytoplasm upon entering the cell through the membrane lesion (Figure 2&#38;3). Quantification of dye fluorescence adjacent to the membrane lesion allows for monitoring the time that it takes for the cell membrane to reseal itself. To exemplify the utility of this method, we use dysferlinopathy patient fibroblasts transfected with GFP-conjugated full-length dysferlin (DYSF) plasmids to assess the rescue of cell membrane repair.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">11320033</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">35mm collagen-coated glass-bottom dishes</td>
			<td style="width:71px;">MatTek</td>
			<td style="width:119px;">P53GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Serum-deprived media</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transfection reagent</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15338100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">FM 4-64 Dye</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">T13320</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Tyrode&#8217;s Salts Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T2397</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Confocal laser scanning microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Chameleon Two-photon laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell membrane repair assay using a two-photon laser microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Healthy human fibroblasts, non-treated dysferlinopathy patient fibroblasts, and patient fibroblasts transfected with a plasmid containing the full-length dysferlin sequence were subjected to two-photon laser wounding to assess membrane resealing ability in real time. Healthy human fibroblast cells displayed low levels of FM 4-64 fluorescence activation following laser wounding and non-treated patient fibroblasts exhibited a high degree of relative fluorescence intensity following injury (...

Watch this Scientific Journal Video about Cell Membrane Repair Assay Using a Two-photon Laser Microscope at JoVE.com

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

The overall goal of this procedure is to quantify plasma membrane repairability in muscular dystrophy patient cells. This method can help answer key questions in muscular dystrophy, such as what is the relationship between a given mutation and membrane resealing kinetics. The main advantage of this technique is that it allows realtime quantification of membrane resealing kinetics in living cells.Begin by culturing fibroblast cells in a T225 flask containing 40 milliliters of growth medium in a carbon dioxide incubator at 37 degrees Celsius. When the culture reaches 70 to 80%confluency, rinse the cells two times with 40 milliliters of PBS per wash, followed by the addition of five milliliters of 0.05%trypsin for five minutes at 37 degrees Celsius. When the cells have detached, stop the reaction with 40 milliliters of fresh growth medium and seed two milliliters of cells at a 100, 000 cells-per-milliliter concentration into 35-millimeter collagen-coated glass-bottom plates for an overnight incubation at 37 degrees Celsius and 5%carbon dioxide.Add 150 microliters of fresh serum-deprived medium to one 1.5-milliliter tube per transfection culture. Next add three microliters of transfection reagent to each tube, and incubate the tubes for at least five minutes at room temperature. While the tubes are incubating, add 175 microliters of serum-derived medium and 3.5 micrograms of dysferlin plasmid DNA to an additional 1.5-milliliter tube per plate.Then combine 150 microliters of the plasmid DNA solution with 150 microliters of the transfection reagent into one new tube per plate for a 20-minute incubation at room temperature. The next morning, replace the growth medium with two milliliters of serum-derived medium. At the end of the incubation, evenly distribute the entire 300 microliters of plasmid solution across each plate, and incubate the plates for 24 hours in the carbon dioxide incubator at 37 degrees Celsius.The next day, replace the plasmid supernatant with fresh growth medium and return the plates to the incubator for another 24 hours. To prepare the cells for a two-photon wounding assay, rinse the transfected cultures one time with one milliliter of Tyrode's solution. Then add one milliliter of fresh Tyrode's solution supplemented with one microliter of 2.5-millimolar FM4-64 dye to the cells.Next, using an inverted confocal microscope, create a 0.2 by two micron target in the microscope's software, and place the target at the edge of the cell membrane so that the target line overlaps and lies perpendicular to the cell membrane. Select a 543-nanometer helium neon laser to excite the FM dye signal, and a 488-nanometer argon laser to excite the GFP signal, and set the detection range for 600 to 760 nanometers and 500 to 550 nanometers respectively. Then create a five-minute time series of sequential image scans.To generate a membrane lesion, use a two-photon laser to 820 nanometers at 15%laser power with 10 iterations to bleach the cells 25 seconds after the beginning of the time series. To quantify the FM4-64 dye fluorescence, use the software to draw a six by six micron region of interest, and place the region of interest adjacent to the wounding location within the cell. Then subtract the background value and divide the net increase by the fluorescence value at time zero to calculate the relative fluorescence values for each time point.Healthy human fibroblast cells display low levels of FM4-64 fluorescence activation following laser wounding while non-treated patient fibroblasts exhibit a high degree of relative fluorescence intensity following injury. Patient cells that were transfected with full-length dysferlin plasmid demonstrate a reduced relative FM4-64 fluorescence intensity compared to non-treated patient controls but similar to the fluorescence values observed in healthy controls. While attempting this procedure, it's important to remember the cells will slowly die in the buffer solution, so work quickly and change to fresh cells at regular intervals.After watching this video, you should have a good understanding of how to regenerate the membrane lesions using a two-photon microscope and how to quantify membrane resealing in realtime.

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JoVE Journal

Immunology and Infection

12.1K Görüntüleme.  University of Alberta Faculty of Medicine and Dentistry. The overall goal of this procedure is to quantify plasma membrane repairability in muscular dystrophy patient cells. This method can help answer key questions in muscular dystrophy, such as what is the relationship between a given mutation and membrane resealing kinetics. The main advantage of this technique is that it allows realtime quantification of membrane resealing kinetics in living cells.Begin by culturing fibroblast cells in a T225 flask containing 40 milliliters of growth med...