Name: Single Fiber Isolation Assay for the Assessment of Oxidative Myofiber Behavior (Video) | JoVE Journal
Uploaded: 2026-03-31T13:00:00.000+00:00
Duration: 7 min 1 s
Description: Learn single fiber isolation from murine soleus muscle using optimized collagenase digestion and mechanical trituration. This method yields 300–500 intact myofibers for immunolabeling and pure satellite cell expansion.

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5 minutes

This study was supported by the establishment of university fellowships towards the creation of science technology innovation (JST, grant no. JPMJFS2139 to T.H.) and grants from the Japan Society for the Promotion of Science (KAKENHI 23H00457 awarded to N.L.F., 20J12849 to Y.Mi., 24K22254 to Y. Ma). This study was also supported by Tokyo Metropolitan Government Advanced Research Grant [R2-2] to Y.Ma., N.L.F., and the TMU strategic research fund for innovative research project [R3] to N.L.F. Thanks to Yasuro Furuichi for his advice and useful comments.

All animals were housed in the animal facility of Tokyo Metropolitan University (Permit Numbers: A4-4, A5-9, A6-7, A7-018). All animals were maintained under specific pathogen free (SPF) conditions. Animal handling and experimental procedures employed in this study strictly adhered to the guidelines of the Safety and Ethics Committee of Tokyo Metropolitan University.
1. Reagent Preparation
<ol>
	<li>Wear gloves throughout. In a 25 mL centrifuge tube, prepare 0.5% of Collagenase type I solution in high glucose Dulbecco&#8217;s Modified Eagle Medium (DMEM) + GlutaMAX with 1% penicillin-streptomycin-amphotericin B suspension (AB). After collagenase is well dissolved (lightly tap the tube if needed), sterilize the solution by filtering through a 0.22 &#181;m low-protein-binding filter. 
	NOTE: 2.5 mL of collagenase solution is enough for two soleus (one mouse).</li>
	<li>Prepare 10 mL of 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Heat-inactivate at 60 &#176;C for 30 min, then filter the solution through a 0.22 &#181;m low-protein-binding filter. 
	NOTE: The 5% BSA in PBS solution can be stored at 4 &#176;C for up to 10 days.</li>
</ol>
2. Dissection of the soleus muscles
<ol>
	<li>Layout equipment for muscle dissection (Figure 1A).</li>
	<li>Anesthetize C57BL/6 mice by intraperitoneal injection of a triple anesthetic mixture (0.5 mL/g body weight) containing 1 mg/mL medetomidine hydrochloride, 5 mg/mL midazolam, and 5 mg/mL butorphanol tartrate, then euthanize the mice by cervical dislocation.</li>
	<li>Make sequential skin incisions on the hindlimb and peel away the skin. Remove the surface fascia.</li>
	<li>Separate the plantaris (PLA) tendon from the gastrocnemius (GAS) and soleus (SOL) tendon bundles. Gently remove the PLA by pulling from its tendon (Figure 1B).</li>
	<li>Cut the GAS and SOL tendon bundles and expose the muscles. Insert a pin into the gap between the proximal tendon of the GAS and SOL (Figure 1C).</li>
	<li>Carefully slide the needle toward the distal end to separate SOL and GAS muscle (Figure 1D). Place a second needle adjacent to the ankle joint to stabilize the hindlimb and prevent movement during dissection.</li>
	<li>Cut the tendon bundles from the GAS side (Figure 1E).</li>
	<li>Rest the SOL on the GAS. Carefully adjust the position of the SOL to expose the proximal tendon and cut it with a pair of micro scissors (Figure 1F).</li>
	<li>Position the SOL on the dissection table and remove the remaining tendon (Figure 1G). 
	NOTE: This step is optional; the remaining tendon does not affect the outcome.</li>
</ol>
3. Digestion of the soleus muscles
<ol>
	<li>Transfer the intact soleus into a 25 mL centrifuge tube containing the prepared 0.5% collagenase solution. Incubate at 37 &#176;C for 130 min. 
	NOTE: Every 15&#8211;20 min, one can manually agitate the sample to slightly separate fibers and support collagenase activity.</li>
</ol>
4. Isolation of single fibers under a stereomicroscope
<ol>
	<li>Cut two Pasteur pipettes to create one with a wide bore and one with a narrow opening (Figure 2A). Under flame, sterilize and smooth the holes to avoid muscle damage during trituration.</li>
	<li>To prevent fiber adhesion during isolation, coat Pasteur pipettes and three 50 mm Petri dishes with 5% BSA/PBS. 
	NOTE: A stereomicroscope (Figure 2B) was used throughout the isolation steps.</li>
	<li>After muscle digestion, discard the excess collagenase solution and transfer the soleus into the first dish containing DMEM with 1% AB (Figure 2C).</li>
	<li>Using the wide-bore Pasteur pipette, gently rinse up and down to dissociate fibers (Figure 2D).</li>
	<li>With the narrow-bore Pasteur pipette, transfer intact single fibers along the muscle fiber length to the second dish. Take the opportunity here to remove debris and/or shrunken fibers (Figure 2E,F).</li>
	<li>Transfer high-quality (full-length/intact) single fibers to a third dish to ensure purity with the narrow-bore Pasteur pipette (Figure 2G,H).</li>
	<li>Collect the fibers into a BSA-coated 25 mL centrifuge tube. Gently stir the suspension, then leave it for 5 min at room temperature to allow the fibers to precipitate to the bottom of the tube.</li>
	<li>Using the narrow-bore Pasteur pipette, carefully discard the supernatant, removing any remaining debris. Leave approximately 500 &#181;L of the DMEM with 1% AB solution. This precipitate will contain a highly purified population of single muscle fibers (Figure 2G,H). 
	NOTE: This technique can yield &#62; 300 single fibers, which can then be used for protein and mRNA extraction and subsequent analysis.</li>
</ol>
5. Establishment of highly purified myoblast cultures from soleus fibers
<ol>
	<li>Dilute Matrigel Matrix to 0.1 mg/mL and use it to coat a 100 mm culture dish. Incubate the dish at 37 &#176;C for 30&#8211;60 min until the coating polymerizes evenly.</li>
	<li>Add an equal volume of Accutase to the muscle fibers suspension and incubate at room temperature for 10 min. Subsequently, add 10 mL of growth medium (composed of glucose-free DMEM containing 30% fetal bovine serum, 1% GlutaMAX, 1% chicken-embryo extract, CEE, 1% AB, and 0.01 &#181;g/mL basic fibroblast growth factor, bFGF)44. 
	NOTE: An Equal amount of Accutase in this case is the 500 &#181;L described in Section 4.8. Accutase facilitates the gentle dissociation of muscle fibers and helps release satellite cells, supporting the subsequent expansion of the myoblast culture.</li>
	<li>Transfer the 10 mL of Accutase-treated muscle fiber suspension into a Matrigel-coated dish. Approximately 4 single fibers/cm2 are added to a 100 mm culture dish. Ensure the fibers are evenly spread throughout the culture dish.</li>
	<li>After 3 days in culture medium, myoblast colonies can be observed. Replace the growth medium daily until myoblasts reach the required population. Normally, by day 6, myoblast cultures become confluent enough for the experimental phase (Figure 3A).</li>
	<li>Detach myoblasts from their original culture dish using Trypsin and incubate the sample at 37 &#176;C for 2 min. Using a hemocytometer, count the cells and seed according to the well size and the cell confluency required for myogenic progression. 
	NOTE: For a 100 mm dish, add 1 mL of trypsin.</li>
</ol>

<ol><li>Janssen, I., Heymsfield, S. B., Wang, Z. M., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+mass+and+distribution+in+468+men+and+women+aged+18%E2%80%9388+yr%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Skeletal muscle mass and distribution in 468 men and women aged 18–88 yr.</a> J Appl Physiol. 89 (1), 81-88 (2000).</li>
<li>Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Satellite+cell+of+skeletal+muscle+fibers%5BTitle%5D)+AND+%22J+Biophys+Biochem+Cytol%22%5BJournal%5D)">Satellite cell of skeletal muscle fibers.</a> J Biophys Biochem Cytol. 9 (2), 493-495 (1961).</li>
<li>Zammit, P. S., Partridge, T. A., Yablonka-Reuveni, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+skeletal+muscle+satellite+cell%3A+The+stem+cell+that+came+in+from+the+cold%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">The skeletal muscle satellite cell: The stem cell that came in from the cold.</a> J Histochem Cytochem. 54 (11), 1177-1191 (2006).</li>
<li>Zammit, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pax7+and+myogenic+progression+in+skeletal+muscle+satellite+cells%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Pax7 and myogenic progression in skeletal muscle satellite cells.</a> J Cell Sci. 119 (9), 1824-1832 (2006).</li>
<li>Seale, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pax7+is+required+for+the+specification+of+myogenic+satellite+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Pax7 is required for the specification of myogenic satellite cells.</a> Cell. 102 (6), 777-786 (2000).</li>
<li>Günther, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myf5-positive+satellite+cells+contribute+to+Pax7-dependent+long-term+maintenance+of+adult+muscle+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells.</a> Cell Stem Cell. 13 (5), 590-601 (2013).</li>
<li>Gnocchi, V. F., White, R. B., Ono, Y., Ellis, J. A., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Further+characterisation+of+the+molecular+signature+of+quiescent+and+activated+mouse+muscle+satellite+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Further characterisation of the molecular signature of quiescent and activated mouse muscle satellite cells.</a> PLoS One. 4 (4), e5205(2009).</li>
<li>Halevy, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pattern+of+Pax7+expression+during+myogenesis+in+the+posthatch+chicken+establishes+a+model+for+satellite+cell+differentiation+and+renewal%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Pattern of Pax7 expression during myogenesis in the posthatch chicken establishes a model for satellite cell differentiation and renewal.</a> Dev Dyn. 231 (3), 489-502 (2004).</li>
<li>Biressi, S., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+in+the+muscle+satellite+cell+population%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">Heterogeneity in the muscle satellite cell population.</a> Semin Cell Dev Biol. 21 (8), 845-854 (2010).</li>
<li>Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((All+muscle+satellite+cells+are+equal%2C+but+are+some+more+equal+than+others%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">All muscle satellite cells are equal, but are some more equal than others.</a> J Cell Sci. 121 (18), 2975-2982 (2008).</li>
<li>Ortuste Quiroga, H. P., Fujimaki, S., Ono, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pax7+reporter+mouse+models%3A+A+pocket+guide+for+satellite+cell+research%5BTitle%5D)+AND+%22Eur+J+Transl+Myol%22%5BJournal%5D)">Pax7 reporter mouse models: A pocket guide for satellite cell research.</a> Eur J Transl Myol. 33 (4), (2023).</li>
<li>Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiber+types+in+mammalian+skeletal+muscles%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Fiber types in mammalian skeletal muscles.</a> Physiol Rev. 91 (4), 1447-1531 (2011).</li>
<li>Pette, D., Staron, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+isoforms%2C+muscle+fiber+types%2C+and+transitions%5BTitle%5D)+AND+%22Microsc+Res+Tech%22%5BJournal%5D)">Myosin isoforms, muscle fiber types, and transitions.</a> Microsc Res Tech. 50 (6), 500-509 (2000).</li>
<li>Ahmetov, I. I., Vinogradova, O. L., Williams, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+polymorphisms+and+fiber-type+composition+of+human+skeletal+muscle%5BTitle%5D)+AND+%22Int+J+Sport+Nutr+Exerc+Metab%22%5BJournal%5D)">Gene polymorphisms and fiber-type composition of human skeletal muscle.</a> Int J Sport Nutr Exerc Metab. 22 (4), 292-303 (2012).</li>
<li>Guth, L., Samaha, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Qualitative+differences+between+actomyosin+ATPase+of+slow+and+fast+mammalian+muscle%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle.</a> Exp Neurol. 25 (1), 138-152 (1969).</li>
<li>Aguilera-Alcala, N., Morales-Reyes, Z., Martin-Lopez, B., Moleon, M., Sanchez-Zapata, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+scavengers+in+providing+non-material+contributions+to+people%5BTitle%5D)+AND+%22Ecol+Indic%22%5BJournal%5D)">Role of scavengers in providing non-material contributions to people.</a> Ecol Indic. 117, 11(2020).</li>
<li>Sullivan, S. M., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+between+mitochondrial+volume+density+and+capillarity+in+hamster+muscles%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Relationship between mitochondrial volume density and capillarity in hamster muscles.</a> Am J Physiol. 252 (1), H149-H155 (1987).</li>
<li>Fujita, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+lipid+metabolism+and+capillary+density+of+the+skeletal+muscle+following+low-intensity+exercise+training+in+a+rat+model+of+obesity+with+hyperinsulinemia%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Changes in lipid metabolism and capillary density of the skeletal muscle following low-intensity exercise training in a rat model of obesity with hyperinsulinemia.</a> PLoS One. 13 (5), e0196895(2018).</li>
<li>Narusawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slow+myosin+in+developing+rat+skeletal+muscle%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Slow myosin in developing rat skeletal muscle.</a> J Cell Biol. 104 (3), 447-459 (1987).</li>
<li>Faerman, A., Shani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+expression+of+the+regulatory+myosin+light+chain+2+gene+during+mouse+embryogenesis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The expression of the regulatory myosin light chain 2 gene during mouse embryogenesis.</a> Development. 118 (3), 919-929 (1993).</li>
<li>Denardi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+2x-myosin+heavy+chain+is+coded+by+a+muscle+fiber+type-specific+and+developmentally+regulated+gene%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Type 2x-myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene.</a> J Cell Biol. 123 (4), 823-835 (1993).</li>
<li>Ciciliot, S., Rossi, A. C., Dyar, K. A., Blaauw, B., Schiaffino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+type+and+fiber+type+specificity+in+muscle+wasting%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">Muscle type and fiber type specificity in muscle wasting.</a> Int J Biochem Cell Biol. 45 (10), 2191-2199 (2013).</li>
<li>Talbot, J., Maves, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+fiber+type%3A+Using+insights+from+muscle+developmental+biology+to+dissect+targets+for+susceptibility+and+resistance+to+muscle+disease%5BTitle%5D)+AND+%22Wiley+Interdiscip+Rev+Dev+Biol%22%5BJournal%5D)">Skeletal muscle fiber type: Using insights from muscle developmental biology to dissect targets for susceptibility and resistance to muscle disease.</a> Wiley Interdiscip Rev Dev Biol. 5 (4), 518-534 (2016).</li>
<li>Celegato, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parallel+protein+and+transcript+profiles+of+FSHD+patient+muscles+correlate+to+the+D4Z4+arrangement+and+reveal+a+common+impairment+of+slow+to+fast+fibre+differentiation+and+a+general+deregulation+of+MyoD-dependent+genes%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Parallel protein and transcript profiles of FSHD patient muscles correlate to the D4Z4 arrangement and reveal a common impairment of slow to fast fibre differentiation and a general deregulation of MyoD-dependent genes.</a> Proteomics. 6 (19), 5303-5321 (2006).</li>
<li>Ganassi, M., Muntoni, F., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+and+identifying+satellite+cell-opathies+within+muscular+dystrophies+and+myopathies%5BTitle%5D)+AND+%22Exp+Cell+Res%22%5BJournal%5D)">Defining and identifying satellite cell-opathies within muscular dystrophies and myopathies.</a> Exp Cell Res. 411 (1), 112906(2022).</li>
<li>Gallagher, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+84-days+of+bedrest+and+resistance+training+on+single+muscle+fibre+myosin+heavy+chain+distribution+in+human+vastus+lateralis+and+soleus+muscles%5BTitle%5D)+AND+%22Acta+Physiol+Scand%22%5BJournal%5D)">Effects of 84-days of bedrest and resistance training on single muscle fibre myosin heavy chain distribution in human vastus lateralis and soleus muscles.</a> Acta Physiol Scand. 185 (1), 61-69 (2005).</li>
<li>Grimby, G., Broberg, C., Krotkiewska, I., Krotkiewski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+fiber+composition+in+patients+with+traumatic+cord+lesion%5BTitle%5D)+AND+%22Scand+J+Rehabil+Med%22%5BJournal%5D)">Muscle fiber composition in patients with traumatic cord lesion.</a> Scand J Rehabil Med. 8 (1), 37-42 (1976).</li>
<li>Shenkman, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+slow+to+fast%3A+Hypogravity-induced+remodeling+of+muscle+fiber+myosin+phenotype%5BTitle%5D)+AND+%22Acta+Naturae%22%5BJournal%5D)">From slow to fast: Hypogravity-induced remodeling of muscle fiber myosin phenotype.</a> Acta Naturae. 8 (4), 47-59 (2016).</li>
<li>Ohira, T., Kawano, F., Goto, K., Ohira, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responses+of+skeletal+muscles+to+gravitational+unloading+and%2For+reloading%5BTitle%5D)+AND+%22J+Physiol+Sci%22%5BJournal%5D)">Responses of skeletal muscles to gravitational unloading and/or reloading.</a> J Physiol Sci. 65 (4), 293-310 (2015).</li>
<li>Higashino, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+changes+in+muscle+atrophy+and+muscle+fiber+type+conversion+after+spinal+cord+transection+and+peripheral+nerve+transection+in+rats%5BTitle%5D)+AND+%22J+Neuroeng+Rehabil%22%5BJournal%5D)">Early changes in muscle atrophy and muscle fiber type conversion after spinal cord transection and peripheral nerve transection in rats.</a> J Neuroeng Rehabil. 10, 46(2013).</li>
<li>Webster, C., Silberstein, L., Hays, A. P., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+muscle+fibers+are+preferentially+affected+in+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy.</a> Cell. 52 (4), 503-513 (1988).</li>
<li>Pedemonte, M., Sandri, C., Schiaffino, S., Minetti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+decrease+of+IIx+myosin+heavy+chain+transcripts+in+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Early decrease of IIx myosin heavy chain transcripts in Duchenne muscular dystrophy.</a> Biochem Biophys Res Commun. 255 (2), 466-469 (1999).</li>
<li>Rafael, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dystrophin+and+utrophin+influence+fiber+type+composition+and+post-synaptic+membrane+structure%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure.</a> Hum Mol Genet. 9 (9), 1357-1367 (2000).</li>
<li>Kotelnikova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+approach+to+meta-analysis+of+microarray+datasets+reveals+muscle+remodeling-related+drug+targets+and+biomarkers+in+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22PLoS+Comput+Biol%22%5BJournal%5D)">Novel approach to meta-analysis of microarray datasets reveals muscle remodeling-related drug targets and biomarkers in Duchenne muscular dystrophy.</a> PLoS Comput Biol. 8 (2), e1002365(2012).</li>
<li>Banerji, C. R. S., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathomechanisms+and+biomarkers+in+facioscapulohumeral+muscular+dystrophy%3A+Roles+of+DUX4+and+Pax7%5BTitle%5D)+AND+%22EMBO+Mol+Med%22%5BJournal%5D)">Pathomechanisms and biomarkers in facioscapulohumeral muscular dystrophy: Roles of DUX4 and Pax7.</a> EMBO Mol Med. 13 (8), e13695(2021).</li>
<li>Lassche, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sarcomeric+dysfunction+contributes+to+muscle+weakness+in+facioscapulohumeral+muscular+dystrophy%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy.</a> Neurology. 80 (8), 733-737 (2013).</li>
<li>Bischoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proliferation+of+muscle+satellite+cells+on+intact+myofibers+in+culture%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Proliferation of muscle satellite cells on intact myofibers in culture.</a> Dev Biol. 115 (1), 129-139 (1986).</li>
<li>Rosenblatt, J. D., Lunt, A. I., Parry, D. J., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culturing+satellite+cells+from+living+single+muscle+fiber+explants%5BTitle%5D)+AND+%22In+Vitro+Cell+Dev+Biol+Anim%22%5BJournal%5D)">Culturing satellite cells from living single muscle fiber explants.</a> In Vitro Cell Dev Biol Anim. 31 (10), 773-779 (1995).</li>
<li>Collins, C. A., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+grafting+of+single+muscle+fibres%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation and grafting of single muscle fibres.</a> Methods Mol Biol. 482, 319-330 (2009).</li>
<li>Ravenscroft, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissociated+flexor+digitorum+brevis+myofiber+culture+system%E2%80%94a+more+mature+muscle+culture+system%5BTitle%5D)+AND+%22Cell+Motil+Cytoskeleton%22%5BJournal%5D)">Dissociated flexor digitorum brevis myofiber culture system—a more mature muscle culture system.</a> Cell Motil Cytoskeleton. 64 (10), 727-738 (2007).</li>
<li>Keire, P., Shearer, A., Shefer, G., Yablonka-Reuveni, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+skeletal+muscle+myofibers+as+a+means+to+analyze+satellite+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells.</a> Methods Mol Biol. 946, 431-468 (2013).</li>
<li>Smith, L. R., Meyer, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+explants%3A+Ex-vivo+models+to+study+cellular+behavior+in+a+complex+tissue+environment%5BTitle%5D)+AND+%22Connect+Tissue+Res%22%5BJournal%5D)">Skeletal muscle explants: Ex-vivo models to study cellular behavior in a complex tissue environment.</a> Connect Tissue Res. 61 (3-4), 248-261 (2020).</li>
<li>Tarpey, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+and+utilization+of+the+flexor+digitorum+brevis+for+assessing+skeletal+muscle+function%5BTitle%5D)+AND+%22Skeletal+Muscle%22%5BJournal%5D)">Characterization and utilization of the flexor digitorum brevis for assessing skeletal muscle function.</a> Skeletal Muscle. 8 (1), 14(2018).</li>
<li>Omairi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+dystrophin-associated+glycoprotein+complex+composition+by+the+metabolic+properties+of+muscle+fibres%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Regulation of the dystrophin-associated glycoprotein complex composition by the metabolic properties of muscle fibres.</a> Sci Rep. 9 (1), 2770(2019).</li>
<li>Moyle, L. A., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+culture+and+immunostaining+of+skeletal+muscle+fibres+to+study+myogenic+progression+in+satellite+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation, culture and immunostaining of skeletal muscle fibres to study myogenic progression in satellite cells.</a> Methods Mol Biol. 1210, 63-78 (2014).</li>
<li>Hettige, P., Tahir, U., Nishikawa, K. C., Gage, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+the+transcriptomes+of+EDL%2C+psoas%2C+and+soleus+muscles+from+mice%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Comparative analysis of the transcriptomes of EDL, psoas, and soleus muscles from mice.</a> BMC Genomics. 21 (1), 808(2020).</li>
<li>Blackburn, D. M., Lazure, F., Soleimani, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+sequencing+of+single+myofibers+from+Mus+musculus%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">RNA sequencing of single myofibers from Mus musculus.</a> Bio Protoc. 10 (4), e3525(2020).</li>
</ol>

The authors have no conflicts of interest to declare.

The first key to isolating a large number of muscle fibers from the soleus (SOL) is to maintain the structural integrity of the muscle during dissection. If the SOL&#8217;s epimysium is torn or if the muscle is pulled forcefully, especially along its longitudinal axis, during dissection, the isolation yield will be severely compromised. These dissection errors are usually easy to identify afterward. Indeed, after collagenase digestion, the muscle becomes uniformly shrunken and forms a white, compact mass. Under such conditions, only a very small number of viable fibers can be dissociated.
The second decisive factor for successful soleus isolation is the efficiency of the type I collagenase digestion. When collagenase is working optimally, approximately 100&#8211;200 soleus fibers can be obtained during the first 10 rinse sessions. In contrast, when digestion is suboptimal, even after the first 20 rinses, less than 50 fibers may be released. To obtain a higher fiber yield, more vigorous rinsing and additional rinse cycles are required. However, because SOL fibers are exceptionally fragile, this often results in fiber shrinkage during rinsing.
For establishing a pure SOL-derived primary myoblast culture, collagenase digestion again plays a crucial role. When digestion is efficient, fewer fibers remain attached to residual tissue and transferring the fibers into culture medium results in minimal fibroblast contamination. In contrast, when the SOL is poorly digested, dissociated fibers will remain attached to residual tissue, contaminating the myoblast culture with fibroblasts. Researchers should look for the typical signs of fibroblast contamination in the seeded cell samples; these include much larger cell size and extensive cytoskeletal protrusions, compared to the much smaller, spindle-shaped satellite cells in adhered Matrigel-coated dishes. Should contamination occur, researchers may subject cells to pre-plating steps as described elsewhere45. Briefly, SCs are cultured for 3 days post-isolation, then trypsinized and briefly pre-plated on collagen-coated dishes. During 15&#8211;30 min of incubation, fibroblasts adhere while SCs remain suspended and are transferred to fresh Matrigel-coated plates.
As mentioned above and consistent with our experience, SOL fibers are longer and more fragile than fibers from the extensor digitorum longus (EDL) and flexor digitorum brevis (FDB). This makes transferring fibers using Pasteur pipettes challenging. It is essential to aspirate fibers along their longitudinal axis rather than drawing them from the middle; otherwise, the fibers fold and shrink under the stress. Aside from the decisive factors that directly determine whether fiber isolation succeeds, a few additional technical steps are essential for improving soleus fiber yield. One of these is to coat all Pasteur pipettes, dishes, and collection tubes with 5% BSA. This prevents freshly dissociated muscle fibers from adhering to surfaces, thereby preventing fiber folding and subsequent shrinkage. It is recommended to prepare new heat-sterilised, BSA-coated Pasteur pipettes each time muscle samples are isolated. Alternatively, researchers could prepare a set of Pasteur pipettes for long-term use. If so, heat-sterilise each time and freshly coat with BSA before use.
The conventional protocol for single muscle fiber isolation was primarily optimized for glycolytic muscles such as FDB and EDL37. However, oxidative muscles, such as the SOL, are structurally distinct. Transcriptomic analyses have shown that extracellular matrix (ECM)-related gene expression differs between the SOL and EDL46, including genes such as dystrophin, a cytoskeletal linker protein of skeletal muscle44. Consequently, the standard condition for EDL muscle digestion with 0.2% (2 mg/mL) collagenase for 60&#8211;90 min is not optimal for SOL muscles, resulting in compact fiber bundles or shrunken fibers after digestion. In this study, we optimized several key steps to adapt the protocol for oxidative SOL muscles. By adjusting the type I collagenase concentration to 0.5% (5 mg/mL), extending the incubation period to 130 min, and minimising mechanical stress during rinsing, 300&#8211;500 intact fibers from a single mouse can be consistently obtained. Compared to EDL, the fiber number and yield of SOL samples remain less (the EDL can yield &#62; 500 fibers); nevertheless, the fiber number and, importantly, the high quality of our SOL-derived fibers are robust and suitable for downstream applications such as immunolabelling and immunoblotting. Although our immunoblotting results consisted of pooled single fiber samples, the technique presented in this report is able to reliably extract intact individual fibers, making them suitable for downstream applications such as immunoblotting, proteomics, and RNA-sequencing47 at single-fiber resolution. Researchers in the field can use this technique to study metabolic and contractile differences between oxidative and glycolytic muscle fibers.
Overall, this protocol provides a reproducible and accessible method for isolating intact oxidative fibers from the soleus muscle, expanding the toolkit available for investigating muscle fiber type-specific physiology and molecular mechanisms.

Skeletal muscle is the most abundant tissue in the human body, accounting for approximately 40&#8211;50% of total body mass1. Skeletal muscle serves as a reservoir of energy in the form of glycogen. During cold exposure, it provides heat through thermogenesis and importantly provides locomotion. Skeletal muscle is composed of multinucleated, post-mitotic myofibers. To sustain muscle maturation and repair, new myonuclei are generated by our skeletal muscle&#39;s resident stem cell, the satellite cell (SC). SCs are found on the periphery of postnatal skeletal muscle fibers2,3,4. SCs are identified by the transcription factor Pax7, which is expressed in both quiescent and activated states3,5. Upon injury or hypertrophic stimulation, SCs activate and upregulate MyoD, enter the myogenic program, proliferate, and ultimately fuse with existing or newly forming myofibers to restore muscle function6,7,8. SCs are heterogeneous in their stemness ability, embryological origin (somitic vs non-somitic), stage of postnatal growth (young vs old), and muscle composition (fast vs slow fibers)9,10,11.
Depending on the region of the body, skeletal muscle is tailored to meet the energetic and functional demands of that region. This heterogeneity in muscle fibers is possible due to their contractility and oxidative capacity. Indeed, muscle fibers are composed of slow-twitch oxidative fibers (termed type I and IIa) and fast contracting glycolytic fibers (termed IIx, in rodents IIb)12,13. This beautiful, functional mosaic of fiber types enables us to move freely in our everyday lives. For example, postural muscles such as the soleus (SOL) and erector spinae are in a state of chronic, low-level activation, and thus contain a high proportion of slow-oxidative type-I and type-IIa muscle fibers. In contrast, short but fast-acting muscles such as the extensor digitorum longus (EDL) are mainly composed of glycolytic type-IIx and type-IIb fibers. The fiber composition, however, is not fixed; rather, muscle fibers are dynamic structures capable of shifting from fast-to-slow and vice versa depending on the functional requirements placed upon them. The difference in fiber type contractility is possible due to the content of myosin protein. This is the main protein within skeletal muscle, composed of isoforms of myosin light and heavy chains12,14.
Inside a sarcomere (muscle&#8217;s smallest functional unit that generates force), myosin heads and actin interact to form a structure known as a crossbridge. Through hydrolysis of ATP, energy is released by the cycling of the myosin and actin interactions15,16. The Myosin heavy chain (MyHC) is the most appropriate marker for fiber type identification12,13. Depending on the isoform of MyHC expressed, the rate of muscle contractility will vary. As mentioned, type-I fibers are slow oxidative fibers, unlike type-II fibers. Type-I dominant muscle groups contain a higher abundance of oxidative enzymes, are richer in mitochondria, and have a dense capillary network to perfuse the muscle17,18 (think marathon runner). Type-I fibers are encoded by the MYH7 gene13,19. Type-II fibers are fast to fatigue, contain a high proportion of glycolytic enzymes, and have a greater maximum velocity of shortening, making them ideal for short bursts of power or movement (think power lifter). Type-II fibers for type-IIa, IIx, and IIb (in rodents) are encoded by the MYH2, MYH1, and MYH4 genes, respectively20,21. Unsurprisingly, any pathophysiological insult that affects muscle integrity will alter fiber type composition and lead to detrimental effects on muscle function, potentially lowering quality of life. In fact, many muscular dystrophies either start in the satellite cell (satellite cell-opathies) or in the muscle fiber (motor neuron- or muscle tissue-derived), and can cause a shift in fiber type composition22,23,24,25. For example, Spinal cord injury, bedrest studies, and spaceflight (microgravity) show that long-term inactivity can trigger postural muscles to shift from a slow to a fast fiber phenotype26,27,28,29,30. Conversely, in Duchenne muscular dystrophy, both in human and mouse models (mdx), fast glycolytic fibers are affected to a greater extent and with greater severity than their slower counterparts31,32,33,34. Similarly, in facioscapulohumeral muscular dystrophy (FSHD), a fast-to-slow twitch transition is observed with a greater proportion of type-I fibers along with lower force production in type-II fibers24,35,36.
It remains unclear why certain muscle fiber types are selectively affected or spared in different diseases. The fact that these patterns are non-random underscores the importance of dissecting the underlying mechanisms, for which single-fiber isolation has emerged as a powerful and widely used technique. Indeed, since first introduced in the mid-80s by Bischoff37, this indispensable tool has been continually refined and adapted, enabling increasingly precise investigation of muscle biology38,39,40. Importantly, single-fiber isolation maintains SCs as close as possible to their physiological niche, enabling adequate scrutiny of their molecular activity. Although muscle groups such as those from the flexor digitorum brevis (FDB) and EDL can be extracted with relative ease41,42,43, the SOL muscle remains one of the most difficult muscles to obtain a rich population of intact fibers from. This is due to its rigidity, long fiber length, and susceptibility to damage. To address this, a technique has been optimised in the present study that robustly yields a high-quality, viable population of fibers from the notoriously difficult-to-isolate SOL muscle. When combined with EDL fiber isolation, this approach enables culture of fibers and derivative cells (satellite cells, myoblasts, and myotubes) from distinct muscle groups. Critically, this platform allows systematic investigation of the molecular factors that may differentially contribute to diverse muscle pathologies and provides a powerful means to test pharmaceutical interventions in a way that can reveal muscle-type-specific responses and ultimately support the development of effective treatments for skeletal muscle diseases.

<table><tbody><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT104</td><td></td></tr><tr><td>Basic fibroblast growth factor</td><td>Peprotech</td><td>450-33</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9418</td><td></td></tr><tr><td>Butorphanol tartrate</td><td>Meiji Yakuka Co., Ltd</td><td>711-12</td><td>Product name: Vetorphale injection</td></tr><tr><td>Chicken Embryo Extract</td><td>US Biological</td><td>C3999</td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington</td><td>M9B2717</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10437-028</td><td></td></tr><tr><td>Glucose-free DMEM</td><td>Gibco</td><td>A14430-01</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>10569</td><td></td></tr><tr><td>High-glucose DMEM+GlutaMAX</td><td>Gibco</td><td>10569-010</td><td></td></tr><tr><td>Horse serum</td><td>Life technology</td><td>16050-130</td><td></td></tr><tr><td>Low-protein binding filter</td><td>Millipore</td><td>SLGVR33RB</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Medetomidine hydrochloride</td><td>Kyoritsu Seiyaku Co., Ltd</td><td>8816</td><td>Product name: Dorbene Injection</td></tr><tr><td>Midazolam</td><td>Maruishi Pharmaceutical Co., Ltd</td><td>4987211762100</td><td>Product name: Dormicum Injection</td></tr><tr><td>Penicillin-streptomycin-amphotericin B suspension</td><td>Wako</td><td>161-23181</td><td></td></tr><tr><td>Ponceau S stain solution</td><td>Sigma-Aldrich</td><td>24-3875-5</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (25 mL)</td><td>IWAKI</td><td>2362-025</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Keyence</td><td>BZ-X810</td><td></td></tr><tr><td>Pasteur pipettes</td><td>IWAKI</td><td>5 3/4 IK-PAS-5P</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M165C</td><td></td></tr><tr><td>Sterilin dishes (100-mm)</td><td>Thermo Scientific</td><td>3020-100</td><td>Referred as Petri dish in the manuscript</td></tr><tr><td>Anbitodies for immunolabelling</td><td></td><td></td><td></td></tr><tr><td>Goat anti-mouse (H+L), Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-32723</td><td>1:400</td></tr><tr><td>Goat anti-rabbit (H+L), Alexa Fluor 594</td><td>Thermo Fisher Scientific</td><td>A-11012</td><td>1:400</td></tr><tr><td>Mouse Anti-Myosin Heavy Chain</td><td>R&amp;amp;D Systems</td><td>MAB4470</td><td>1:400</td></tr><tr><td>Mouse Anti-Pax7</td><td>DSHB</td><td>AB-528428</td><td>1:100</td></tr><tr><td>Rabbit Anti-MyoD</td><td>Santa Cruz Biotechnology</td><td>sc-760</td><td>1:100</td></tr><tr><td>Anbitodies for immunoblotting</td><td></td><td></td><td></td></tr><tr><td>HRP-conjugated Anti-mouse IgG</td><td>Cytiva</td><td>NA931</td><td>1:1000/ horseradish peroxidase</td></tr><tr><td>HRP-conjugated Anti-Rabbit IgG</td><td>Cytiva</td><td>NA934</td><td>1:1000/ horseradish peroxidase</td></tr><tr><td>Mouse Anti-MyHC I</td><td>Sigma</td><td>M8421</td><td>1:1000/ Myosin Heavy Chain I</td></tr><tr><td>Mouse Anti-MyHC II</td><td>Sigma</td><td>M4276</td><td>1:1000/ Myosin Heavy Chain II</td></tr><tr><td>Mouse Anti-PGC-1&amp;alpha;</td><td>Santa Cruz Biotechnology</td><td>sc-517380</td><td>1:1000/ Pparg coactivator 1 alpha</td></tr><tr><td>Mouse Anti-&amp;alpha;-tubulin</td><td>Cell Signaling Technology</td><td>#3873</td><td>1:1000/ Alpha tubulin</td></tr><tr><td>Rabbit Anti-COXIV</td><td>Cell Signaling Technology</td><td>#4850</td><td>1:1000/ Cytochrome c oxidase IV</td></tr><tr><td>Rabbit Anti-GAPDH</td><td>Cell Signaling Technology</td><td>#2218</td><td>1:10000/ Glyceraldehyde-3-Phosphate Dehydrogenase</td></tr><tr><td>Rabbit Anti-GLUT4</td><td>Cell Signaling Technology</td><td>#2213</td><td>1:1000/ Glucose Transporter 4</td></tr><tr><td>Rabbit Anti-&amp;beta;-actin</td><td>Proteintech</td><td>20536-1-AP</td><td>1:2000/ Nonmuscle Beta-actin</td></tr></tbody></table>

single fiber isolation assay for the assessment of oxidative myofiber behavior

Single Fiber Isolation Assay for the Assessment of Oxidative Myofiber Behavior

Skeletal muscle is composed of multinucleated myofibers whose integrity and function are essential for movement and overall health. In neuromuscular disorders, muscle groups can be affected differentially across disease types and stages, making it important to isolate viable single fibers from representative muscles for mechanistic studies. The objective of this protocol is to provide a reliable method for isolating a high yield of intact single myofibers from the murine soleus (SOL) muscle, which is challenging to dissociate due to fiber length and fragility. By optimizing collagenase concentration and digestion time, the protocol minimizes fiber loss and preserves viability during dissociation. The optimized workflow markedly reduces residual tissue attachment to isolated fibers, thereby limiting carryover of non-fiber cells and minimizing cross-cell contamination. This technique is suitable for single-fiber analyses as well as satellite cell isolation and subsequent expansion under conditions with minimal contamination. Using this optimized workflow, we routinely obtain ~300&#8211;500 intact SOL-derived single myofibers that can be immunolabelled with high efficiency and also yield a high-quality satellite cell population with minimal fibroblasts contamination. Using comparative analyses with Extensor Digitorum Longus (EDL) muscle we underscore the value of a tailored, muscle-specific approach to evaluating therapies in neuromuscular disease research, enabling candidate pharmaceutical interventions to be tested within small, tunable experimental windows.

Our technique demonstrates that isolated fibers of the soleus (SOL) and extensor digitorum longus (EDL) muscles can be immunolabelled efficiently against Pax7 (Figure 3B). These isolated fibers can be kept in a floating culture medium (composed of DMEM + GlutaMAX with 10% horse serum, 1% AB, 0.5% CEE) for 3 days, followed by immunolabelling (Figure 3B). Keeping floating fibers for longer than 4 days is not recommended. Under the described floating fiber conditions, myofibers tend to aggregate after this time, compromising subsequent immunolabelling analysis.
The presented technique can further be used for immunoblotting across the myogenic program (Figure 3C). Indeed, SOL- and EDL-derived myoblasts were plated into 24-well plates at approximately 70% confluence. The cells were either maintained in growth medium for 1 day (myoblasts) or induced to differentiate for two or four days (early-forming and differentiated myotubes, respectively). Five µg of protein was loaded for immunoblotting analysis.
MyHC I expression was almost specifically expressed in SOL-derived muscle fibers, with minimal expression in myotubes and negligible in myoblasts (Figure 3C). MyHC II was expressed in both SOL and EDL muscle fibers. This expression reflects the SOL, composed mainly of type-I and IIa, while the EDL is composed primarily of IIx and IIb fibers. Myoblasts did not express MyHC II, but its expression increased progressively during differentiation. The oxidative-related proteins PGC-1α and COXIV demonstrated the expected higher expression in SOL-derived fibers. Also, a higher expression of GLUT4 was detected in isolated SOL fibers compared to EDL fibers. An issue that skeletal muscle biologists may face is that widely used housekeeping proteins and loading controls, such as α-tubulin, β-actin, GAPDH and total protein stains including Ponceau S do not remain stable across myoblasts, myotubes, and isolated muscle fibers, with each showing distinct variability. The signal intensity of α-tubulin and β-actin was highest in myotubes, while Ponceau S staining was relatively weak in myoblasts. In contrast, GAPDH showed stronger expression in EDL muscle fibers. Nevertheless, depending on the experimental purpose, one of these housekeeping proteins may still be applicable, alternatively normalization factors such as heat shock protein 70 (HSP70) can be explored.
<img alt="Dissection setup showing muscle identification; surgical tools, labeled muscles: SOL, GAS." class="xfigimg" src="/files/ftp_upload/70637/70637fig1.jpg" data-altupdatedat="1784606404000" data-alt="Figure 1"> 
Figure 1. Dissection of soleus muscle. (A) Equipment required for dissection. 1: micro scissors, 2: fine forceps, 3: dissecting scissors, and 4: dissection pins. (B) PLA tendon was cut from GAS and SOL tendon bundles (yellow arrow). The distal PLA (white arrow) tendon was lifted with forceps. (C) A dissection pin was inserted into the space between the proximal tendon of the SOL (red arrow) and GAS (green arrow). (D) SOL was separated from GAS using a dissection pin. (E) GAS and SOL tendon bundles were then cut at the GAS side. (F) SOL proximal tendon was then cut with micro scissors. (G) Additional distal tendon bundles were removed. <a href="https://www.jove.com/files/ftp_upload/70637/70637fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy setup and images; pipettes, microscope, fungal growth under varied magnifications." class="xfigimg" src="/files/ftp_upload/70637/70637fig2.jpg" data-altupdatedat="1784606404000" data-alt="Figure 2"> 
Figure 2. Isolation of soleus fibers. (A) Equipment required for isolation. 1: narrow-bore and 2: wide-bore Pasteur pipettes. (B) Stereomicroscope used for isolation. (C) Collagenase-digested soleus was transferred to DMEM with 1% AB. (D) Soleus fibers dissociated from the muscle after the first 10 rinse sessions. (E) Single fibers transferred to second dish. (F) Image showing clean fibers removed of debris and shrunken fibers. (G) Debris-free single fibers transferred to the third dish. (H) Intact single fibers clearly visible. Scale bar = 10 mm. <a href="https://www.jove.com/files/ftp_upload/70637/70637fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="muscle differentiation; microscopy of SOL/EDL myofibers; Pax7/MyoD staining; Western blot analysis" class="xfigimg" src="/files/ftp_upload/70637/70637fig3.jpg" data-altupdatedat="1784606404000" data-alt="Figure 3"> 
Figure 3. Expansion of cell culture and analysis of EDL and SOL-derived samples. (A) Following 6 days after isolation, the proliferation medium cultured samples show a pure myoblast culture obtained from isolated soleus fibers. Scale bar = 100 µm. (B) SOL and EDL-derived single fibers, immediately or 3 days after isolation, show satellite cells immunolabelled for Pax7 (green). The absence of MyoD (red) confirms that satellite cells have not yet committed to myogenic activation. Fibers were counterstained with DAPI (blue). The cells were subsequently observed through a fluorescence microscope. Scale bar = 20 µm. (C) 5 µg of protein was used for immunoblotting of myoblasts, myotubes, and myofiber derived samples. Immunoblotting panels show expression of MyHC I, MyHC II, PGC-1α, COX IV, GLUT4, α-tubulin, β-actin and GAPDH were used as housekeeping/normalising targets. Ponceau S stain was used as a total protein loading control. <a href="https://www.jove.com/files/ftp_upload/70637/70637fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes the isolation of intact single muscle fibers from the oxidative soleus and the establishment of highly purified myoblast cultures derived from these fibers. The optimized procedure reliably yields approximately 300&#8211;500 intact myofibers per mouse, enabling downstream molecular and cellular analyses.

Skeletal muscle is composed of multinucleated myofibers whose integrity and function are essential for movement and overall health. In neuromuscular ...

This research focuses on the isolation of intact single muscle fibers from the oxidative solute muscle. Existing single fiber isolation methods inadequately preserve fragile long soleus fibers. This optimized protocol overcomes damage, enabling recovery of intact fibers.To begin, lay out the equipment required for muscle dissection. Use the pre-prepared triple anesthetic mixture to sedate the C57 black six mouse by intraperitoneal injection. Then euthanize the animal by cervical dislocation.Make sequential skin incisions on the hind limb and peel away the skin to expose underlying tissue. Then remove the surface fascia. Separate the plantaris tendon from the gastrocnemius and soleus tendon bundles.Gently remove the plantaris by pulling from its tendon. Cut the gastrocnemius and soleus tendon bundles to expose the muscles. Insert a pin into the gap between the proximal tendon of the gastrocnemius and soleus.Carefully slide this needle toward the distal end to separate the soleus and gastrocnemius muscles. Insert a second needle into proximal tendon of the gastrocnemius and soleus. Cut the tendon bundles from the gastrocnemius side.Rest the soleus on the gastrocnemius. Adjust the position of the soleus to expose the proximal tendon. Cut the proximal tendon with micro scissors.Position the soleus on the dissection table. Remove the remaining tendon. Transfer the intact soleus into a 25 milliliter centrifuge tube containing 0.5%collaginase solution.Incubate at 37 degrees Celsius for 130 minutes. Cut two pastor pipettes to create one with a wide bore and one with a narrow opening. Sterilize and smooth the openings under flame to prevent muscle damage during trituration.Use serum solution containing 5%BSA in PBS to coat the pastor pipettes and three 50 millimeter Petri dishes to prevent fiber adhesion. After digestion, discard the excess collagenase solution. Transfer the soleus into a dish containing DMEM with 1%antibiotic antimicotic.Use the wide bore pastor pipette to gently pipette up and down to dissociate fibers. Use the narrow bore pastor pipette to transfer intact single fibers along their length to a second dish. Remove debris and shrunken fibers during transfer.Now transfer high quality intact single fibers to a third dish using the narrow bore pastor pipette. Collect the fibers into a BSA coated 25 milliliter centrifuge tube. Gently stir the suspension and leave it at room temperature for five minutes to allow fibers to precipitate.Use a narrow bore pasture pipette to discard the supernatant, leaving approximately 500 microliters of the medium. Retain the precipitate containing purified single muscle fibers. Dilute Matrigel matrix to a concentration of 0.1 milligrams per milliliter in DMEM.Use it to coat a 100 millimeter culture dish. Incubate the dish at 37 degrees Celsius for 30 to 60 minutes or until polymerization is complete. Add around 500 microliters of accutase to the previously prepared muscle fiber suspension and incubate at room temperature for 10 minutes.Then add 10 milliliters of growth medium containing glucose-free DMEM with supplements. Transfer 10 milliliters of the accutase treated muscle fiber suspension into the matrigel-coated dish. Add approximately four single fibers per square centimeter to the 100 millimeter culture dish.Transfer the fibers evenly throughout the culture dish. Use a microscope to check the even distribution of the fibers. Observe myoblast colonies after three days in culture.Replace the growth medium daily until the cells reach confluence. Continue culture until confluence is achieved by day six. After six days in proliferation medium, isolated soleus fibers yielded a pure myoblast culture.Soleus derived single fibers and extensor digitorum longus derived single fibers immediately after isolation showed satellite cells immunolabeled for paired box seven. Soleus-derived single fibers and extensor digitorum longest derived single fibers cultured for three days in floating culture medium also showed satellite cells immuno-labeled for paired box seven. This protocol enables high resolution analysis of molecular behavior within single fibers while yielding a rich, viable satellite subpopulation.Preserving soleus structure integrity during dissection and precisely optimizing type one collagenase digestion are critical for preventing fiber damage. Future studies can utilize enriched population of solus fibers for comprehensive analysis of molecular mechanisms underlying muscle behavior.

single-fiber-isolation-assay-for-assessment-oxidative-myofiber

Research

JoVE Journal

Biology

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

This video demonstrates the isolation of single myofibres from harvested murine Extensor Digitorum Longus (EDL) muscle. The isolated myofibres can be cultured to study myofiber-associated muscle stem cell activity.

Myofiber Isolation: A Technique to Obtain Single Myofibers from Harvested Mouse Extensor Digitorum Longus (EDL) 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.

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

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

Fiber Type Identification of Human Skeletal Muscle

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Learn single fiber isolation from murine soleus muscle using optimized collagenase digestion and mechanical trituration. This method yields 300–500 intact myofibers for immunolabeling and pure satellite cell expansion.

Single Fiber Isolation Assay for the Assessment of Oxidative Myofiber Behavior

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