1. Treadmill Running for Revealing the Extent of Muscle Injury in Mouse Models 
<ol>
 <li>Establish the angle of the treadmill surface for use during the running protocol. Generally, a flat level or an angle between 7&deg;and 15&deg;degrees downhill or uphill is used. Some treadmills have in integral apparatus to adjust the incline while others require the treadmill to be elevated through other means.</li>
 <li>Place a tray or a blue lab pad underneath the treadmill before putting the animals in the treadmill to collect any waste from the animals during the running protocol.</li>
 <li>Before running mice should be acclimated to the environment of the treadmill. This involves placing the animals in the treadmill for 15 minutes with the electric grid off and the belt drive motor on but the belt not moving (i.e. with the speed set at 0 m/s).</li>
 <li>Turn on the motivational electric grid. The intensity and frequency of the pulses used can usually be controlled on treadmills but varies from one manufacturer to another. Generally, maximum intensity is not required to motivate mice to run while a high frequency (at least once every two seconds) will improve compliance.</li>
 <li>Activate the treadmill belt to begin the running protocol. An initial speed of approximately 5 meters/min should be used for a warm up period for the mice. The speed of the treadmill can be accelerated slowly, usually by adding 1-2 meters/min for each minute following the start of the treadmill.</li>
 <li>Compliance of animals in most protocols can be improved by conducting a series of short runs (5 minutes) at warm up speed for 3 to 10 days before the experimental running begins.</li>
 <li>An acute exhaustion exercise will usually involve increasing the speed of the treadmill with time until a maximum speed (generally &lt; 30 m/m) is achieved and maintained until the mice show signs of exhaustion. Criteria for judging exhaustion vary but generally include a mouse spending more than half of the time on the electric grid or 5 consecutive seconds on the electric grid without returning to the treadmill surface. After an individual mouse is exhausted it can be removed from the treadmill and the total time of running can be recorded.</li>
 <li>Changing the angle of the treadmill can be used in acute running trials to increase the exercise load (with an uphill angle) or to induce damaging eccentric contractions (with a downhill angle) that tear the sarcolemmal membranes of muscle fibers1. Evans blue dye can be injected into animals before such procedures for use as a histological dye to identify damaged muscle fibers2,3.</li>
 <li>Endurance training running protocols provide an exercise load to the animal over a prolonged period of time (up to months) in order to produce remodeling of the muscle or the cardiovascular system. Warm up procedure is the same as above (step 1.5) however the maximum speed is generally lower and the running times can be quite long (&gt;1 hour).</li>
 <li>After completion of running the mice can be returned to their cages. Treadmills usually become quite dirty while the mice are running, particularly over long protocols. It is necessary to clean the treadmill, usually with a 70% ethanol spray, following each use.</li>
</ol>
2. Ex Vivo Assay of Membrane Repair Capacity of Isolated Muscle Fibers Following UV-laser Damage
<ol>
 <li>Dissect out the superficial layer of the flexor digitorum brevis (FDB) muscle from the mouse foot. First, cut the skin of the sole open at the median line while taking care not to damage the muscle underneath, then make horizontal cuts and remove the skin. The flat white tendon of FDB muscle (proximal tendon) can be seen attached to the calcaneus bone. Bluntly separate the tendon by forceps and sever the tendon close to site of attachment, grab the free tendon by forceps and pull it up gently to separate the FDB from surrounding tissues while cutting any connective tissues that remain attached. Upon seeing the distal tendons branch to the individual digits the FDB may be removed by cutting where these tendons connect to the deeper layer of muscle.</li>
 <li>Put the FDB muscle bundle in 1 ml of collagenase solution pre-warmed to 37&deg;C in a 1.5 ml Eppendorf tube, tape the tube in a horizontal orientation in a 37&deg;C orbital shaker and shake at 200 rpm for 65 min. Times of incubation may have to be adjusted to ensure sufficient digestion, which is indicated by a frayed appearance and pale color of the muscle.</li>
 <li>Carefully transfer the digested FDB bundle into a 1.5 &mu;l centrifuge tube containing ~600 &mu;l of 2.5 Ca2+ Tyrode via a 1 ml pipette tip with a cut end that has a diameter sufficient to allow the digested bundle to easily pass through without disruption. Gently flip the tube to shake the loose fibers from the bundle.</li>
 <li>Cut a 30 &mu;L pipette tip so that the diameter is only slightly smaller than the muscle bundle (between 15 - 20 &mu;m in diameter).Use the pipettor to gently draw the bundle into the pipette and then push it back into the solution. Repeat this process until the majority of the fibers are disassociated from the bundle.</li>
 <li>Mix disassociated fibers well by gently turning the tube, take desired amount and drop onto a glass-bottom delta T dish. The volume to load will vary based on the efficiency of isolation and the number of useful fibers that are needed on a dish for a particular protocol. The remaining fibers can be stored in the tube at 4&deg;C for approximately 6 hours for additional studies.</li>
 <li>Allow the dish to sit undisturbed for 5 minutes. This allows the fibers to adhere to the glass bottom of the dish.</li>
 <li>Place the glass bottomed dish on a confocal microscopy equipped with a UV laser. Observe the fibers under phase microscopy at &gt;100X magnification to check for the presence of a normal striation pattern and a straight, rod like shape.</li>
 <li>Add FM1-43 or FM4-64 styrylpyridinium dyes4to solution to a final concentration of 2.5 &mu;M5.</li>
 <li>To induce damage to the fiber position it in the imaging window so the fiber is oriented at a 45&deg; angle from the top left of the field of view to the bottom right (Figure 1). Irradiate a 5x5 pixel area (approximately 0.9&mu;mx0.9&mu;m)of the plasma membrane using a UV laser (80 mW or greater, 351/364 nm) set to maximum power for 5 seconds. The irradiated region should split the plasma membrane; that is, half of the 5x5 box should be inside the fiber, while the remainder should be outside the fiber (Figure 1).</li>
 <li>Capture images of dye entry into the fiber at intervals of 5 seconds each for up to 5 minutes.</li>
 <li>Repeat step 2.8 for no more than 3 fibers per dish, as the fluorescent dye will eventually be endocytosed into the fiber and complicate any data analysis. After 3 fibers a new dish should be prepared from step 2.5.</li>
 <li>Analyze data by calculating the change in fluorescence intensity (&Delta;F/F0) between each captured frame to measure the amount of dye entry. This requires measuring the mean fluorescence of an area approximately 200 &mu;m2 using analysis software such as the publically available ImageJ (http://rsbweb.nih.gov/ij/). For comparison between fibers, the following calculation should be made for each frame: &Delta;F/F0, where F0 is the mean fluorescence of the region of interest in the first captured frame (t = 0; prior to injury), and &Delta;F is the change in fluorescence of each subsequent frame (F-F0).</li>
</ol>
3. Live Cell Confocal Imaging to Monitor the Dynamic Process of Cell Membrane Repair
<ol>
 <li>Micropipettes were made from PYREX Capillaries. The capillary was placed on a micropipette puller and pulled by pre-set program (Heat=695; Pull=50; Vel.=55; Time=250).</li>
 <li>Micropipette was attached to a 3 axis (xyz) micromanipulator.</li>
 <li>Cells are transfected with plasmid DNA using normal methods on a glass bottom delta T dish. Media should be switched to 2.5mM Ca2+ Tyrode buffer before experiments begin.</li>
 <li>Glass dishes containing transfected cells were placed on a laser scanning confocal microscope with 40X 1.3NA oil immersion objective.</li>
 <li>The acute live-cell damage was performed by inserting the micropipette into the cell membrane and then quickly retract the micropipette out of the cell3,6. Consecutive live cell images were obtained at an interval of 1.54 s/frame.</li>
 <li>For the saponin induced membrane disruption assay, 0.005% saponin (Sigma) in 2.5mM Ca2+ Tyrode buffer was perfused by a gravity-flow custom-assembled perfusion system7. The perfusion rate is approximately 1 ml/min and the perfusion tip should be placed directly above the cell being imaged.</li>
</ol>
4. Representative Results:
A representative movie of the mouse treadmill running can be made during filming. The movie will illustrate the reduced running capacity of the mg53-/- mice due to damage to the skeletal muscle.
The extent of damage caused by the UV-laser damage protocol detailed above depends on the membrane repair capacity of the fiber analyzed. This capacity is affected by the genotype of the mouse from which the fiber was isolated, extracellular conditions, and any alterations to protein expression (transfection or infection). A normal wild type fiber will allow slight to moderate dye entry (Figure 2a). Muscle fiber derived from the mg53-/- mice with compromised membrane repair capacity will display more significant entry of dye into the fiber (Figure 2b).
Typical results for microelectrode injury show translocation of MG53-containing vesicles to the injury site. Figure 3a and 3b show GFP-MG53 transfected C2C12 myotubes damaged by insertion of the micropipette into the cell membrane. In the cell before micropipette damage, GFP-MG53 is located on both plasma membrane and cytoplasm3. Following micropipette penetration, GFP-MG53 containing vesicles moved towards damage sites (as indicated by arrowhead). Figure 3c shows a GFP-MG53 transfected C2C12 myoblasts treated with 0.005% of saponin, a detergent that can permeabilize the plasma membrane. Upon saponin treatment, GFP-MG53 translocated from cytosol to the cell membrane.
<img src="/files/ftp_upload/2717/2717fig1.jpg" alt="Figure 1"/> 
Weisleder, et al. Figure 1. Fiber alignment and injury region definition. Proper orientation of the isolated muscle fiber within the field of view and definition of the injury region shown.
<img src="/files/ftp_upload/2717/2717fig2.jpg" alt="Figure 2"/> 
Weisleder, et al. Figure 2. UV laser damage of FDB fiber. Images show fibers prior to injury (0 sec, left) and at 200 sec post injury (right). Fibers from a wild type mouse (a) show slight injury. Fibers from mice with compromised membrane repair (b) allow more dye to enter.
<img src="/files/ftp_upload/2717/2717fig3.jpg" alt="Figure 3"/> 
Weisleder, et al. Figure 3. Microelectrode and saponin damage to cultured cells. Images show C2C12 myotubes (a, b) and a C2C12 myoblast (c) prior to injury (above) and post injury (bottom). Cells injured with a microelectrode (a, b) show MG53 translocation to injury sites (arrows). Cells treated with saponin (c) show MG53 translocation to the cell membrane.

<ol>
	<li>Armstrong, R. B., Ogilvie, R. W., Schwane, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eccentric+exercise-induced+injury+to+rat+skeletal+muscle.">Eccentric exercise-induced injury to rat skeletal muscle.</a> J Appl Physiol. 54, 80-93 (1983).</li><li>Hamer, P. W., McGeachie, J. M., Davies, M. J., Grounds, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evans+Blue+Dye+as+an+in+vivo+marker+of+myofibre+damage:+optimising+parameters+for+detecting+initial+myofibre+membrane+permeability.">Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.</a> J Anat. 200, 69-79 (2002).</li><li>Cai, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MG53+nucleates+assembly+of+cell+membrane+repair+machinery.">MG53 nucleates assembly of cell membrane repair machinery.</a> Nat Cell Biol. 11, 56-64 (2009).</li><li>Cochilla, A. J., Angleson, J. K., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+secretory+membrane+with+FM1-43+fluorescence.">Monitoring secretory membrane with FM1-43 fluorescence.</a> Annu Rev Neurosci. 22, 1-10 (1999).</li><li>McNeil, P. L., Miyake, K., Vogel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endomembrane+requirement+for+cell+surface+repair.">The endomembrane requirement for cell surface repair.</a> Proc Natl Acad Sci U S A. 100, 4592-4597 (2003).</li><li>Cai, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+repair+defects+in+muscular+dystrophy+are+linked+to+altered+interaction+between+MG53,+caveolin-3,+and+dysferlin.">Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.</a> J Biol Chem. 284, 15894-15902 (2009).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotection+of+ischemia/reperfusion+injury+by+cholesterol-dependent+MG53-mediated+membrane+repair.">Cardioprotection of ischemia/reperfusion injury by cholesterol-dependent MG53-mediated membrane repair.</a> Circ Res. 107, 76-83 (2010).</li><li>Knab, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeatability+of+exercise+behaviors+in+mice.">Repeatability of exercise behaviors in mice.</a> Physiol Behav. 98, 433-440 (2009).</li><li>Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+resistance+to+fatigue+and+altered+calcium+handling+properties+of+sarcalumenin+knockout+mice.">Enhanced resistance to fatigue and altered calcium handling properties of sarcalumenin knockout mice.</a> Physiol Genomics. 23, 72-78 (2005).</li><li>Massett, M. P., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+differences+in+responses+to+exercise+training+in+inbred+and+hybrid+mice.">Strain-dependent differences in responses to exercise training in inbred and hybrid mice.</a> Am J Physiol Regul Integr Comp Physiol. 288, 1006-1013 (2005).</li><li>Bansal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li></ol>

No conflicts of interest declared.

Treadmill running is a useful methodology at providing an exercise load to the treated animals. As a methodology for measuring muscle function or endurance capacity it is a notoriously noisy approach that is difficult to execute in a consistently reproducible fashion8. In general, times to exhaustion can be used as an endpoint to resolve major differences between experimental groups, such as those between some knockout mouse lines and wild type mice9. Experimental manipulations that produce more subtle difference, such as some pharmaceutical trials, may not resolve differences above the noise associated with this technique. In order to maximize the reproducibility and sensitivity of these techniques it is important to maintain the conditions for session to session very closely. The time of day the animals are exercised, as well as the warm up period conditions used, are important factors and should stay consistent from trial to trial.
Strain effects can have significant impact on the design of treadmill protocols as certain strains of mice can run for much more time on treadmill than other strains and respond differently to endurance exercise10. As a general guideline, many mice will be able to run 200-300 total meters in an exhaustion study with constant increase in the speed of the treadmill (5 m/m initial speed with 1 m/m added for each minute after start). For an endurance exercise the mice may be run between 9 to 12 m/m for 30 minutes 2 or 3 times a week. However, individual studies will usually require some optimization of the protocol to match individual experimental needs.
The UV-laser damage procedure has been shown to be an effective method for measuring the membrane repair capacity of skeletal muscle fibers3,6,11. One vital component to the success of these experiments is to use only muscle fibers that display a straight, rod shaped appearance with a regular pattern of striations and a smooth sarcolemma that does not appear wrinkled. If other muscle fibers are selected membrane damage may already be present in these fibers and interpretation of results from these experiments may be complicated. It is also important that the orientation of the fiber and definition of the irradiated region be as defined in Figure 1. This provides an injury of reproducible size, thus allowing comparison between fibers. If the defined region of injury lies completely within the fiber, an internal injury will be created rather than disrupting the sarcolemma. Additionally, the value calculated for &Delta;F/F0 is very sensitive to the region of interest selected for measurement of mean fluorescence. An area of approximately 200&mu;m2, adjacent to and including the injury site, is appropriate. Unlike the region defined for injury, this area should lie completely within the fiber. If you include space outside the fiber, the resulting blank area will increase &Delta;F/F0 in fibers with little dye entry while lowering &Delta;F/F0 in fibers with a more severe phenotype. This decreases the sensitivity of the assay, thus making comparison between fibers more difficult.
For the micropipette damage assay, the angle between the micropipette and glass bottom dish should be approximately 45 degree in order to effectively damage the cell membrane. The cells were chosen for micropipette damage should be tightly attached to the bottom of the dish and rounded cells should not be used since most likely they will detach after micropipette poking. Saponin permeabilzation experiments are less technically demanding and relatively quicker to perform compared to micropipette penetration assays. However, there are several limitations to saponin damage, including that only one cell can be used per dish since saponin will diffuse and damage other cells in that dish.

<table border="1">
<tbody>
	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 	<td>Rodent treadmill</td>
 <td>Columbus Instruments Exer 3/6 or equivalent</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>70 % ethanol</td>
 <td>ISC Bioexpress</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Dissection tools including fine tip forceps, spring scissors</td>
 <td>World Precision Instruments</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>2 mg/ml collagenase type I</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>0 Ca2+ Tyrode buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl2, pH 7.2, 290 mosm)</td>
 <td> Sigma</td>
 <td>&nbsp;</td>
 </tr>
	<tr>
 	<td>2.5mM Ca2+ Tyrode buffer (140 mM NaCl, 2.5 mM CaCl2, 5 mM KCl, 10 mM HEPES, 2 mM MgCl2, pH 7.2, 290 mosm)</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 </tr>	
	<tr>
 	<td>Temperature controllable orbital shaker</td>
 <td>New Brunswick or equivalent</td>
 <td>&nbsp;</td>
 </tr>
	<tr>
 	<td>Delta TPG Dish</td>
 <td>Fisher Scientific</td>
 <td>1207133</td>
 </tr>
 <tr>
 	<td>LSM 510 confocal microscope with an Enterprise 80 mW UV laser or equivalent confocal microscope</td>
 <td>Zeiss</td>
 <td>&nbsp;</td>
 </tr> 
	<tr>
 	<td>FM1-43 or FM4-64 dyes</td>
 <td>Invitrogen</td>
 <td> &nbsp; </td>
 </tr>
	<tr>
 	<td>Borosilicate Capillaries Size 0.8-1.0 X 100 mm</td>
 <td>PYREX</td>
 <td>Part No. 9530-2</td>
 </tr>
	<tr>
 	<td>Micropipette puller</td>
 <td>Sutter Instruments</td>
 <td>model P-97</td>
 </tr>
	<tr>
 	<td>3 axis micromanipulator</td>
 <td>Narishige</td>
 <td>MHW-3</td>
 </tr>
	<tr>
 	<td>Radiance 2100 laser scanning confocal microscope or equivalent microscope</td>
 <td>BioRad</td>
 <td>MHW-3</td>
 </tr>
 <tr>
 	<td>Saponin</td>
 <td>Sigma</td>
 <td>47036</td>
 </tr>
 </tbody>
</table>

visualization of mg53-mediated cell membrane repair using in vivo and in vitro systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Watch this Scientific Journal Video about Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems at JoVE.com

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

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12.3K ビュー.  Robert Wood Johnson Medical School. 動物全体でMG53を介した細胞膜修復のと細胞​​レベルで動的なプロセスを視覚化するために使用されるプロトコルは、ここで説明する。これらのメソッドは、細胞膜の再シール性と再生医療の細胞生物学を調査するために適用することができます。

ビデオ: Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.