We would like to thank Sarah Dick for providing critical reading and comments. M.A.R. holds the Canada Research Chair in Molecular Genetics and is an International Research Scholar of the Howard Hughes Medical Institute. This work was supported by grants to MAR from the National Institutes of Health, the Howard Hughes Medical Institute, the Canadian Institutes of Health Research, the Muscular Dystrophy Association and the Canada Research Chair Program.

All experiments were handled according to the University of Ottawa regulations for animal care and handling.
See Table 1 for an overview of the protocol.
1. Before Starting the Isolation
<ol>
 <li>Prepare the following solutions: 
 0.2% Collagenase type I in DMEM (Dulbecco's modified Eagle's medium; high glucose, L-glutamine with 110 mg/ml sodium pyruvate). For two EDL muscles prepare 2 ml of 0.2% Collagenease in DMEM. Filter the solution through 0.22 &mu;m filter. 10 min before the isolation, prewarm at 37 &deg;C in a water bath. Additional aliquots of undiluted collagenase can be frozen at -20 &deg;C for later use. 
 Note: use DMEM with sodium pyruvate throughout all of the procedure. Fibers do not survive in sodium pyruvate-free medium.
 <ul>
 <li>Washing media (use to perform all the washes). Supplement DMEM with 1% Penicillin/Streptomycin. Filter through 0.22 &mu;m filter before use.</li>
 <li>Myofiber culture media. Supplement DMEM with 20% FBS, 1% Chicken Embryo extract and 1% Penicillin/Streptomycin. Filter through 0.22 &mu;m filter before use.</li>
 <li>Matrigel coated dishes. To culture fibers for long period of time, we recommend using Matrigel as coating substrate. To prepare Matrigel coated dishes, thaw an aliquot of Matrigel at 4 &deg;C overnight. The day after, dilute Matrigel 1:10 in DMEM. Keep Matrigel at 4 &deg;C at all times and avoid abrupt temperature changes as this will create microscopic crystals within the Matrigel solution resulting in uneven coating. Place dishes to be coated on ice. Coat dishes with just enough volume to cover the surface. Let it sit for 1 min. Completely remove the leftover Matrigel. Let dishes dry at 37 &deg;C for at least 3 hr prior to use or overnight.</li>
 </ul>
 
 Note: aliquots of diluted Matrigel can be re-used multiple times for coating purpose if stored at 4 &deg;C.</li>
 <li>Lastly, be sure to clean the microscope station and dissecting tools with 70% ethanol.</li>
 <li>For two EDL isolations (one mouse) prepare five plastic Petri dishes (60*15mm) as follows:
 <ul>
 <li>Four dishes for the isolation (1 for muscle dissociation, 3 for serial washes). All dishes must be coated with horse serum (HS) to prevent myofibers from attaching to plastic. To coat, pipette 3 ml of horse serum into each dish, swirl to allow even coating, remove the horse serum and let the dish dry for at least 30 min. Add 4 ml of DMEM to each dish. Keep dishes at 37 &deg;C in a 5% CO2 incubator prior to use.</li>
 </ul>
 
 Note: Horse serum can be re-used multiple times, if kept sterile. Alternatively, a solution of 10% HS in DMEM can be used to coat dishes. Use HS coated dishes throughout the whole protocol and when culturing myofibers on floating conditions.
 <ul>
 <li>One dish will be used to culture myofibers after the isolation. Alternative dish sizes or formats can be used for the final culture of myofibers depending on downstream applications. However, we do not suggest dish sizes larger than 60*15mm. Myofibers can be kept in suspension for no longer than 96 hr before hyper contraction occurs. For longer culture times, we recommend using Matrigel coated dishes or plates.</li>
 </ul>
 </li>
 <li>For one fiber isolation (2 EDL), prepare two sterile Pasteur pipettes: one large bore pipette for muscle handling and one small bore pipette for myofiber manipulation. Use a diamond pen to cut each glass pipette to the desired length and heat polish to smooth pipette's edges. By using the flame, curve the tip of the small bore pipette. This will help handling single fibers. Flame to sterilize. Coat each pipette with HS before use.</li>
</ol>
2. Muscle Dissection and Digestion
<ol>
 <li>For the purpose of these experiments, 8 week old SV129, Pax7 CreERCklr;Rosa26TdTomato (Pax7Cre-TdTomato) and Myf5-Cre;Rosa26YFP were used.</li>
 <li>Spray hind limbs with 70% ethanol. Pin the animal (face up) to a support board to have a better grasp of the hind limb during the procedure.</li>
 <li>With the help of scissors, cut through the entire length of the limb and expose the underlying muscle. Remove the skin as well as any hair or fur (Figure 1A).</li>
 <li>With a fine scissor, cut through the thin fascia without damaging the underlying muscles. Visually localize the EDL. The EDL is found in the anterior compartment of the hind limb just underneath the Tibialis Anterior (TA) muscle.</li>
 <li>With the help of two forceps, expose the distal tendons.</li>
 <li>Cut the distal tendons (of both the TA and EDL) with sharp Cohann-Vannas spring scissors.</li>
 <li>With the help of forceps hold both TA and EDL muscles by their tendons and delicately pull the muscles up towards the proximal end. At this point you should be able to clearly see the EDL muscle just underneath the TA muscle. Now, separate the EDL from the TA muscle by pulling the two tendons in opposite directions (Figure 1B). Avoid stretching the EDL muscle while performing this operation as this will damage the myofibers.</li>
 <li>Expose the EDL tendon. To better visualize the proximal tendon it may help at this point to remove the TA muscle. It may also help to cut off some the connective tissue around the knee (Figure 1C).</li>
 <li>Cut the proximal tendon and gently remove the EDL (Figure 1D).</li>
 <li>By holding the muscle through its tendons, transfer it to 2 ml of previously prepared collagenase solution. Incubate at 37 &deg;C in a water bath. 
 Note: for successful fiber isolation, it is important to isolate the EDL from tendon to tendon so that the myofiber integrity is maintained. Steps 2.3 to 2.9 can be performed under a dissecting microscope. Alternatively, the use of a magnifying glass may also help to have a better view of the muscles.</li>
 <li>Repeat steps 2.3 to 2.9 to isolate the second EDL. Transfer the second EDL in the same tube. To avoid uneven digestion, isolation of the second EDL should be completed no more than 5 min after the first. 
 Note 1: incubation time may need to be adjusted depending on collagenase activity. Longer or shorter incubation might be required depending on the size, age and/or muscle condition (e.g. fibrotic muscles need longer digestion time). 
 Note 2: If examining satellite cells behavior under quiescence conditions, agitation during muscle digestion time may activate satellite cells 10.</li>
 <li>During the digestion time, regularly check the muscle to avoid over-digestion. Stop the digestion when muscles start to loosen up and myofibers are visible. To stop digestion, carefully transfer both muscles to a prewarmed Petri dish with 4 ml of DMEM (dissociation dish). Use the large bore size pipette to perform this operation. 
 Note: avoid muscle overdigestion as this inevitably results in the isolation of hyper contracted myofibers.</li>
</ol>
3. Single Myofiber Dissociation and Culture
<ol>
 <li>To release myofibers, use the large bore glass pipette to flush the muscle with warm medium until fibers naturally start being released. Do not triturate the muscle as this will inevitably result in damaging fibers. Perform this and the following steps under a dissecting microscope.</li>
 <li>Continue releasing myofibers until the desired number is reached. If the dish is required at room temperature for more than 10 min allow a 5 min (minimum) incubation at 37 &deg;C, 5% CO2 to re-equilibrate the medium. 
 Note: if medium reaches temperatures below physiological (37 &deg;C) for an extended time myofibers will die.</li>
 <li>Using the small size bore pipette, transfer live single myofibers to a new prewarmed dish (first of three consecutive washes). Handle each myofiber individually instead of transferring bulk of myofibers all at once. If necessary, incubate at 37 &deg;C, 5% CO2 for 10-15 min to re-equilibrate the medium.</li>
 <li>Repeat step 3.3 for 2 more times or until all dead myofibers and debris are removed. We recommend at least three consecutive washes for proper clean-up. 
 Note: dead myofibers will appear as short and hyper contracted under the microscope light.</li>
 <li>Incubate single myofibers at 37 &deg;C, 5% CO2 in the last wash dish (DMEM only) for at least one hr prior to switching to culture medium. This allows myofibers to adjust to the in vitro conditions in the absence of serum. We found that immediate culture of myofibers in serum rich medium increases fiber shrinking.</li>
 <li>After one hr, transfer myofibers to a new prewarmed dish or to the appropriate culture format depending on the downstream application. Culture fibers in high serum medium to allow satellite cell activation. Alternatively, different concentrations of serum or chicken embryo extract can also be used. Change medium every other day.</li>
</ol>
4. Downstream Applications
<ol>
 <li>Immunostaining. Live myofibers can be fixed and stained at any time point during the isolation. Immunofluorescence can be performed on both floating and substrate-attached myofibers. If staining floating myofibers, use a small bore glass pipette to transfer myofibers from one solution to another. Alternatively, it is possible to keep myofibers in the same well throughout all the procedure and add/remove solutions using a glass pipette. Avoid standard aspiration as this will result in the removal of the myofibers as well. Briefly, completely remove culture medium; fix myofibers in prewarmed 4% paraformaldehyde (PFA) for 5 min. Extensively wash in PBS several times. Incubate myofibers in 1% glycine in PBS or other standard quenching solutions to minimize PFA background staining. If necessary, permeabilize myofibers with 0.1% Triton X-100 in PBS for 10 min followed by a 5 min wash in PBS. Incubate fibers in blocking solution (10% horse serum, 0.1% Triton X-100, 1% NaN) for 1 hr at room temperature or preferably overnight at 4 &deg;C. Alternative blocking solutions may be used, depending on the antibody of interest. Wash once in PBS for 5 min. Incubate with the appropriate primary antibody diluted in blocking solution for 1 hr at room temperature or overnight at 4 &deg;C. Wash fibers 3 times at 5 min per wash in PBS to remove any unbound antibody. Incubate with the appropriate secondary antibody for 45 min to 1 hr at room temperature. Wash 3 times at 5 min per wash in PBS. Counterstain nuclei with 1 mg/ml DAPI. If staining floating fibers, transfer each fiber to a glass slide suitable for microscopy. Remove any excess of PBS or medium. Apply mounting medium and then add coverslip. Proceed to visualize myofibers under a fluorescence microscope. See Figure 4 for representative immunofluorescence on EDL myofibers. Alternative staining procedures using stronger fixatives are described in Verma, M. et al. 11 and Wosniak, A.C. et al. 12.</li>
 <li>Oligonucleotide or plasmid transient transfection. Live myofibers can be transfected with plasmid or siRNA for specific gene/s of interest. When using siRNA, double transfection is recommended for efficient gene knockdown. We suggest performing the first transfection after 8 hr from the isolation. Six hr after the transfection, replace with fresh medium. For siRNA transfection, we suggest starting by using a final concentration of 50 nM. Gene expression knockdown can be analyzed by RNA extraction or preferably by immunostaining.</li>
 <li>Viral infection. Infection of live myofibers is possible although the incidence of cell death is higher than with oligo transfection and the efficiency of infection may be variable depending on muscle conditions and age. For instance, intact adult muscle myofibers are particularly non responsive to viral infection due to the presence of the basal lamina which has been shown to provide a protective barrier against host infection 13, 14. Lentiviral vectors are preferred over retroviral vectors because they can infect quiescent (non mitotic) cells. For viral infection, it is advisable to culture fibers on a Matrigel coated dish or plate and let myofibers adjust to media conditions for the first 24 hr.</li>
 <li>Live Imaging. Live imaging of myofibers is particularly time consuming and necessitates a microscope equipped with a 37 &deg;C, 5% CO2 chamber. It is useful when assessing the behavior of single satellite cells. Work done using this technique has been instrumental for the discovery of satellite cell heterogeneity and to study their behavior on fibers (15 and 16).</li>
</ol>

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C., Misunoya, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+muscle+fiber+isolation+and+culture+for+cellular+molecular,+pharmacological,+and+evolutionary+studies.">Single muscle fiber isolation and culture for cellular molecular, pharmacological, and evolutionary studies.</a> Methods Mol. Biol. 798, 85-102 (2012).</li><li>Shefer, G., Yablonka-Reuveni, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+skeletal+muscle+myofibers+as+a+means+to+analyze+satellite+cells.">Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells.</a> Methods Mol. Biol. 290, 281-304 (2005).</li><li>White, R. B., Bi&#233;rinx, A. S., Gnocchi, V. F., Zammit, P. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+muscle+stem+cells+by+fluorescence+activated+cell+sorting+cytometry.">Isolation of muscle stem cells by fluorescence activated cell sorting cytometry.</a> Methods Mol. Biol. 798, 53-64 (2012).</li><li>Engler, A. D., Griffin, M. A., Sen, S., Bonnemann, C. G., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myotubes+differentiate+optimally+on+substrates+with+tissue-like+stiffness:+pathological+implications+for+soft+or+stiff+microenvironments.">Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments.</a> J. Cell. Biol. 166 (4), 877-887 (2004).</li><li>Boonen, K. J., Post, M. 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G., Watkin, S. C., Hoffman, E. P., Rosenblatt, J. D., Glorioso, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basal+lamina+is+a+physical+barrier+to+herpes+simplex+virus-mediated+gene+delivery+to+mature+muscle+fibers.">The basal lamina is a physical barrier to herpes simplex virus-mediated gene delivery to mature muscle fibers.</a> J. Virol. 70 (11), 8117-8123 (1996).</li><li>Feero, W. G., Rosenblatt, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+gene+delivery+to+skeletal+muscle:+insights+on+maturation-dependent+loss+of+fiber+infectivity+for+adenovirus+and+herpes+simplex+type+1+viral+vectors.">Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.</a> Hum. Gene Ther. 8 (4), 371-380 (1997).</li><li>Kuang, S., Kuroda, K., grand, F. L. e., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+self-renewal+and+commitment+of+satellite+stem+cells+in+muscle.">Asymmetric self-renewal and commitment of satellite stem cells in muscle.</a> Cell. 129 (5), 999-1010 (2007).</li><li>Siegel, A. L., Atchison, K., Fisher, K. E., Davis, G. E., Cornelison, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+timelapse+analysis+of+muscle+satellite+cell+motility.">3D timelapse analysis of muscle satellite cell motility.</a> Stem Cells. 27 (10), 2527-2538 (2009).</li><li>Seale, P., Sabourin, L. A., Girgis-Gabardo, A., Mansouri, A., Gruss, P., Rudnicki, M. 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No competing interests.

The isolation and culture of single myofibers from intact muscles provides an excellent in vitro model to study the process of muscle regeneration. A unique feature of this system is the preservation of satellite cells in their physiological environment beneath the basal lamina. Most importantly, the technique can be used to investigate muscle stem cell behavior in both quiescence and activated states. Over the past 20 years, the myofiber culture system has provided meaningful insights into the biology of the satellite cell population with respect to both intrinsic and extrinsic determinants. By culturing individual myofibers, satellite cell heterogeneity with respect to myofiber, muscle type or regenerative potential has been addressed 15. Elaborate studies using live imaging of single cultured fibers allowed for the gaining information on satellite cell migration pattern 16. The acquisition of quantitative data is also possible. Although time consuming, it provides significant information on the distribution and occurrence of specific events (stem cells asymmetric division, proliferation and differentiation ratio, etc.). Together with previously described applications, protein or RNA extraction from single fibers is also possible, although isolation of pure population of satellite cells by FACS or other means may provide a better platform to investigate protein or gene expression changes at a molecular level. In our experience, the most critical step for successful fiber isolation is the "tendon to tendon" isolation (steps 2.5 to 2.9 in the protocol text). This guarantees that after muscle digestion, fibers are released from the EDL with minimal or no damage thus increasing their performance in the following steps.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma-Aldrich</td>
 <td>C0130-1g</td>
 <td>Prepare fresh all times for optimal results. Additional aliquots of undiluted collagenase can be stored at -20 &deg;C.</td>
 </tr>
 <tr>
 <td>DMEM High Glucose + NaPyr </td>
 <td>Invitrogen</td>
 <td>11995073</td>
 <td>Addition of Pen/Strep is necessary to minimize air contaminations</td>
 </tr>
 <tr>
 <td>Characterized Fetal Bovine Serum</td>
 <td>Hyclone</td>
 <td>SH30396.03</td>
 <td>Lot #KUJ35152</td>
 </tr>
 <tr>
 <td>Horse Serum</td>
 <td>Hyclone</td>
 <td>SH300.74.03</td>
 <td>Lot #AVJ82494</td>
 </tr>
 <tr>
 <td>Chicken Embryo Extract</td>
 <td>Accurate Chemical</td>
 <td>CE-650-T, Batch B7035010</td>
 <td>Avoid freezing-thawing cycles. </td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD</td>
 <td></td>
 <td>Store aliquots at -20 &deg;C. Avoid freezing-thawing cycles. Store diluted aliquots at 4 &deg;C.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Cohann-Vannas Spring Scissor 6mm Blades-Straight-Sharp</td>
 <td>Fine Science Tools</td>
 <td>15000-02</td>
 <td></td>
 </tr>
 <tr>
 <td>Extra Thin Iris Scissors- 10.5cm</td>
 <td>Fine Science Tools</td>
 <td>14088-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Moria-Iris Forceps Curved</td>
 <td>Fine Science Tools</td>
 <td>11373-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Pasteur Pipettes</td>
 <td>VWR</td>
 <td>14672-380</td>
 <td>14672-200</td>
 </tr>
 <tr>
 <td>Diamond Pen</td>
 <td>VWR</td>
 <td>52865-005</td>
 <td></td>
 </tr>
 <tr>
 <td>60*15mm Petri Dish</td>
 <td>VWR</td>
 <td>25384-092</td>
 <td></td>
 </tr>
 <tr>
 <td>Acrodisc 0.22 &mu;m Filter</td>
 <td>VWR</td>
 <td>CA28-145-477</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting Microscope StemiV6</td>
 <td>Zeiss</td>
 <td></td>
 <td>An additional light source may be required to have a better focus view</td>
 </tr>
 </tbody>
</table>

isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Here we described the isolation of single myofibers from the EDL muscle of adult mice. Successful myofibers yield depend on several factors, such as collagenase activity or muscle conditions or age. Most importantly, the isolation of the muscle from tendon to tendon results in long, intact myofibers from EDL muscle. Figure 1 shows a step by step graphical representation of the "tendon to tendon" isolation. Once the muscle is fully digested, myofibers are released by applying gentle pressure to the muscle. Figure 2A shows a representative picture of a myofiber isolation experiment after the first wash. At this point the culture contains a mixture of bundles of single myofibers which appear as long and shining tubular structures, hyper contracted myofibers which are dark and short and debris from the digestion process. By the end of at least three consecutive washes, only single live myofibers should remain in the dish for culture or downstream analysis (Figure 2B). Figure 2C shows a long, live fiber as compared to a hyper contracted fiber (C'). At the time of the isolation, satellite cells appear as tiny protuberances on the myofiber surface (Figure 3A). If maintained in serum rich medium, satellite cells activate, proliferate, migrate and eventually fuse into myotubes (B). After 15 days in culture, all myotubes express the Myf5 reporter YFP (C) suggesting that muscle regeneration in the adult is performed by cells that at some point during their development had expressed the myogenic determinant factor Myf5. All satellite cells express the paired box transcription factor Pax7 17. The reporter mouse line Pax7Cre-TdTomato can be used to trace satellite cells via the expression of the TdTomato fluorescent signal (Figures 3D and E). Figure 4 shows a representative immunofluorescence of double Pax7+/Myod satellite cells. Classically double Pax7+/Myod+ satellite cells are considered proliferative committed muscle progenitors which will either complete the differentiation program by down regulating Pax7 while maintaining Myod expression or return to quiescence by down regulating Myod and maintaining Pax7 expression 18
<img src="/files/ftp_upload/50074/50074fig1.jpg" alt="Figure 1"/> 
 Figure 1. EDL muscle isolation from mouse hindlimb. A . Hindlimb of an 8 week old adult mouse. Arrows indicates the distal (knee) tendon and the proximal (foot) tendon. B. Anatomical position of the Tibialis Anterior (TA) muscle and the Extensor Digitorum Longus (EDL) muscle. C. By holding the EDL through the proximal tendon, the muscle is pulled toward the knee to expose the distal tendon. D. The distal tendon is cut and the EDL is released.
<img src="/files/ftp_upload/50074/50074fig2.jpg" alt="Figure 2"/> 
 Figure 2. Representative results of a single myofiber isolation experiment. A . Bright field picture of a myofiber isolation experiment at the first wash step. Red arrow indicates live myofibers, white arrow indicates hyper contracted myofibers and yellow arrow indicates cells, myofiber or ECM debris. B. After consecutive washes in DMEM, only live myofibers remain. C and C'. Image represents a long intact live myofiber (C) and a short hyper contracted myofiber (C'). Bar 10 &mu;m. Pictures were taken with a Zeiss Axio Observer Z1 microscope equipped with AxioCam HR.
<img src="/files/ftp_upload/50074/50074fig3.jpg" alt="Figure 3"/> 
 Figure 3. Representative results of a single myofiber culture experiment. A . Bright field picture of a single, live myofiber with its associated satellite cell (arrow) right after the isolation (Time 0). B. Single myofibers were plated on Matrigel and cultured in serum rich medium for 15 days. White arrow indicates differentiated myotubes as compared to single cells (yellow arrow) C. Single myofibers from a Myf5Cre;RosaYFP mouse were cultured as in B. Arrow indicates Myf5 derived myotubes. D and E. Single myofibers from Pax7Cre;TdTomato were isolated and fixed right after. Arrows indicate Pax7 positive quiescent satellite cell (E, red). Nuclei were counterstained with DAPI (D). Bar: 50 &mu;m. Pictures were taken with a Zeiss Axio Observer Z1 microscope equipped with AxioCam HR.
<img src="/files/ftp_upload/50074/50074fig4.jpg" alt="Figure 4"/> 
 Figure 4. Example of immunofluorescence staining of satellite cells on myofibers. Single myofibers were isolated and cultured for 72 hr in floating conditions. Satellite cells on myofibers were stained for the satellite cell specific marker Pax7 (B, red) and the myogenic regulatory factor Myod (C, green). Nuclei were counterstained with DAPI (A). Bars: 50 &mu;m. Pictures were taken with a Zeiss Axio Observer Z1 microscope equipped with AxioCam HR.
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Muscle Dissection (5 min each muscle)</td>
 <td><ul>
 <li>Tendon to tendon isolation</li>
 <li>Sharp tools</li>
 <li>Minimal muscle damage</li>
 </ul></td>
 </tr>
 <tr>
 <td>Muscle Digestion (30-45 min)</td>
 <td><ul>
 <li>Temperature sensitive</li>
 <li>Avoid overdigestion</li>
 </ul></td>
 </tr>
 <tr>
 <td>Fiber isolation</td>
 <td><ul>
 <li>Avoid over manipulation of fibers</li>
 <li>Eliminate debris with several washes</li>
 </ul></td>
 </tr>
 <tr>
 <td>Fiber Culture (Up to 3-4 weeks)</td>
 <td><ul>
 <li>Format: any</li>
 <li>Coating: horse serum, Matrigel</li>
 <li>Medium: basal or high serum</li>
 </ul></td>
 </tr>
 </tbody>
</table>
Table 1. Overview of the myofiber isolation protocol. The myofiber isolation protocol consists of 4 major steps. For each step, the approximate time and major critical points are discussed. For detailed discussion of each step, refer to the protocol text.

Watch this Scientific Journal Video about Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle at JoVE.com

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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62.1K Views.  Ottawa Hospital Research Institute. Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Video: Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

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.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...