The authors are grateful to Dr. Matthew Watt at the University of Melbourne and Dr. Anastasia Kralli at Johns Hopkins University for assistance adopting this protocol based on the work of Mokbel et al.6. We also thank Dr. Sabine Jordan for assistance developing and adopting this protocol in our laboratory. This work was funded by the National Institutes of Health R01s DK097164 and DK112927 to K.A.L.

This protocol follows the animal care guidelines of Scripps Research.
1. Collection and Processing of Muscle Tissue Explants
<ol>
	<li>The day prior to dissection, sterilize all dissection equipment (forceps, razor blades, and scissors) and prepare all required media: phosphate buffered saline (PBS), MB Plating media (12.5 mL of DMEM, 12.5 mL of HAMS F12, 20 mL of heat-inactivated fetal bovine serum (FBS), 5 mL of amniotic fluid medium supplement), and Coating Solution (24 mL of DMEM, 24 mL of HAMS F12, 1.7 mL of collagen, 1 mL of matrigel).</li>
	<li>The day of dissection, coat one 6-cm dish with Coating Solution for each muscle to be dissected. Add 2 mL of Coating Solution to the surface of each plate, shake gently to create an even coat on the surface, and incubate the plates with solution at 4 &#176;C for 1 h.</li>
	<li>Remove Coating Solution from the plates and return to the stock solution. 
	NOTE: Coating Solution can be reused for up to six months and should be stored at 4 &#176;C.</li>
	<li>Rinse the plates twice with 2 mL of PBS to remove unbound collagen and matrigel.</li>
	<li>Place the plates in a 37 &#176;C tissue culture incubator during dissections.</li>
	<li>Prepare a moist chamber by placing 2-3 sheets of thick absorbent paper into a plastic bag or a sterile 15 cm dish and use a pipette to wet the surface of the paper with sterile water.</li>
	<li>Place the chamber under UV light for 5 min to sterilize.</li>
	<li>Dissect desired muscles from a 4 to 8-week old mouse. To sterilize muscle tissue, rinse gently in PBS containing 40 &#956;g/mL gentamicin. 
	NOTE: For quadriceps and gastrocnemius muscles, plate one muscle per plate. For soleus, plantaris and EDL, combine muscles from both legs in one plate.</li>
	<li>Use sterile forceps to transfer the muscle to a sterile 10 cm non-coated Petri dish. Add 0.5-1.0 mL of Plating media over the muscle such that the tissue is moist but not floating (Figure 1A).</li>
	<li>Use a sterile scalpel or razor blade to gently slice the muscle into small fragments (approximately 1-3 mm3). 
	NOTE: It is important to minimize handling the muscle tissue for best results.</li>
	<li>Use forceps or a pipette to transfer the muscle fragments onto the surface of a pre-coated 6-cm plate. Very gently overlay an additional 0.8 mL of Plating Media over the tissue. There should be enough media to keep the tissue pieces hydrated but not floating (Figure 1B).</li>
	<li>Place the 6-cm dishes containing the muscle fragments inside the moist chamber and return to an incubator (37 &#176;C, 5% CO2) for 48 h (Figure 1C). 
	NOTE: It is vital that muscle fragments adhere to the surface of the plate to allow for myoblast outgrowth. Do not move the plates/chamber for at least 48 h.</li>
	<li>After 48 h, carefully check the plates to ensure that muscle fragments have adhered. Overlay with 2 mL of Plating Media, taking care not to dislodge the fragments. 
	NOTE: If there is visible contamination or debris on the plates, carefully wash the muscle pieces. Plate 2 mL of PBS/gentamicin solution at the edge of the plate and tip gently to wash over the muscle tissue. Remove the PBS and repeat the wash step. Following the second wash, overlay 2 mL of Plating Media.</li>
	<li>Keep the plates in the 37 &#176;C incubator for up to an additional 3 days (5 days from original dissection) before harvesting myoblasts. Check every other day for outgrowth of myoblasts. 
	NOTE: The appearance of cells emerging from muscle explants will be variable and heterogeneous. Primary myoblasts appear as small, round, and bright cells. However, it is neither necessary nor reliable to identify them by their appearance at this stage (Figure 2).</li>
</ol>
2. Harvesting Outgrowing Myoblasts
<ol>
	<li>Prepare and pre-warm Myoblast Media (17.5 mL of DMEM, 17.5 mL of HAMS F12, 10 mL of FBS, 5 mL of amniotic fluid medium supplement), trypsin, and PBS/gentamicin. Coat one T25 flask per muscle group being harvested with Coating Solution and as described in step 1.2 (see Table 1).</li>
	<li>Remove Plating Media and gently rinse the muscle explants with 2 mL of PBS/gentamicin. Quickly remove PBS (rinse one plate at a time and do not let the plate sit in PBS at this step).</li>
	<li>Gently add 1 mL of PBS/gentamicin and place the plate in the 37 &#176;C incubator for 1 min.</li>
	<li>Use a P1000 pipette to collect PBS/cells in a 15 mL centrifuge tube.</li>
	<li>Add 1 mL of trypsin to the plate and return to the 37 &#176;C incubator for 3 min. Gently tap the plates to dislodge myoblasts. Collect the trypsin/cells and combine with the PBS collection. Add 8 mL of Myoblast Media to the centrifuge tube and gently invert to mix.</li>
	<li>Gently overlay 2 mL of Plating Solution on the muscle plates and return to the 37 &#176;C incubator.</li>
	<li>Spin the centrifuge tubes containing cells in a centrifuge for 3 min at 200 x g.</li>
	<li>Aspirate the supernatant to ~1 mL, being careful to avoid cell pellet. Gently add Myoblast Media (see Table 1) and transfer the cells to the pre-coated flask and place in the 37 &#176;C incubator. This is the P0 harvest. If observed under a microscope, there may be very few cells (Figure 3).</li>
	<li>Repeat the harvest described above every other day up to three times. After the third harvest, discard the explants.</li>
</ol>
3. Expansion and Enrichment of Proliferating Myoblasts
NOTE: The P0 harvest will be heterogeneous (~60% myoblasts). The next 2 passages use PBS to selectively harvest myoblasts. Many of the more adherent cells will be left behind and the rapidly proliferating myoblasts will be &#8805;95% pure within 2 passages. Once myoblasts are established, they should be maintained at a low density to avoid spontaneous differentiation.
<ol>
	<li>For each T25 flask of ~40-50% confluent cells from section 2, coat one T75 flask with 5 mL of Coating Solution and place at 4 &#176;C for 1-4 h.</li>
	<li>Remove Coating Solution from the flasks and return to stock solution. Rinse the flasks twice with 2 mL of PBS/gentamicin and place in the 37 &#176;C incubator.</li>
	<li>Aspirate the media from P0 myoblast T25 flasks. Rinse the cells briefly with 2 mL of warm PBS/gentamicin. Aspirate PBS from the flask. 
	NOTE: The purpose of this step (3.3) is to reduce the possibility of bacterial contamination. If performed quickly and gently, it should not result in loss of myoblasts. It can be omitted to maximize myoblast preservation if desired.</li>
	<li>Pipette 2 mL of warm PBS (not trypsin) into each flask containing myoblasts. Place the flasks with PBS into the 37 &#176;C incubator for 3 min. 
	NOTE: Myoblasts should easily detach from flasks with PBS. Using PBS rather than trypsin at this step is critical for reducing contamination of the myoblast population with other cell types.</li>
	<li>Remove the cells from the 37 &#176;C incubator and firmly tap side of the flasks to dislodge the cells. Check under a light microscope for freely floating myoblasts.</li>
	<li>Place the flasks upright in a tissue culture hood and rinse the bottom of the flasks with 10 mL of Myoblast Media 2-3 times to ensure all cells are dislodged.</li>
	<li>Collect the cell/media mixture in a 15 mL centrifuge tube. Centrifuge for 3 min at 200 x g.</li>
	<li>Aspirate the media to around 1 mL, being careful to avoid the cell pellet. Gently add an appropriate volume of Myoblast Media to centrifuge tube and gently mix.</li>
	<li>Distribute the cell mixture to new T75 flasks. Add 10 mL of Myoblast Media to each new T75 flask. Gently shake the flasks horizontally to distribute the cells and place in the 37 &#176;C incubator overnight.</li>
	<li>Two days later, passage once more with PBS, splitting each T75 flask into three T75 flasks. 
	NOTE: Do not allow myoblasts to become more than 50%-60% confluent, as this would cause them to start differentiating and lose prolifaterive capacity.</li>
	<li>For additional passages, use trypsin. Passaging twice with PBS yields &#62;95% myoblasts; attempts to further improve the purity tends to result in poorer differentiation.</li>
</ol>
4. Differentiation of Primary Myoblasts to Myotubes
<ol>
	<li>Plate P2 (or later passage) myoblasts in Myoblast Media on coated plates (see Table 1 for suggested coating and plating volumes and cell numbers). Two or three days later, when cells are at 70-80% confluency, change the media to Differentiation Media (24 mL of DMEM, 24 mL of HAMS F12, 1.5 mL of heat inactivated horse serum, 0.5 mL of Insulin-Selenium-Transferrin).</li>
	<li>Change Differentiation Media every other day during differentiation of primary myoblasts into myotubes. Differentiation is typically complete by Day 4-5 and will be marked by elongated, fused cells that spontaneously twitch. Perform experiments on differentiated myotubes within 6 days. Typically cells are assayed five or six days after initiating differentiation.</li>
</ol>
5. Measuring Oxygen Consumption Rate in Myoblasts or Myotubes in 96-well Plates
<ol>
	<li>Coat 96-well plates with 25 &#956;L of coating solution per well. Centrifuge the plates at 58 x g for 1 min to remove any bubbles.</li>
	<li>Incubate the plates in coating solution for 1-4 h at 4 &#176;C. Use multi-channel pipette to remove the coating solution and wash three times in 25 &#956;L of cold PBS/gentamicin. Centrifuge at least one of these washes to ensure that no bubbles are trapped.</li>
	<li>Add 40 &#956;L of Differentiation Media to each well. Centrifuge the media on the coated plate with no cells for 1 min at 58 x g to avoid bubbles and remove surface tension to get a uniform layer of media.</li>
	<li>Add cells suspended in an additional 40 &#956;L of media to each well. Spin again at 58 x g. 
	NOTE: Consistency in plating is important for best results and the number of cells plated per well should be optimized for each experimental setup, and will likely be in the range of ~10,000-25,000 myoblasts plated in differentiation media per well of a 96-well plate.</li>
	<li>Gently change the media daily during differentiation. Do not aspirate the media but rather remove it with a pipette, leaving a small volume behind to avoid dislodging the cells or exposing them to the air. For example, replace 50 &#956;L at a time for three repeats rather than all 80 &#956;L at once.</li>
	<li>Use 8-15 wells per condition. It may be necessary to omit some wells if cells do not form a uniform layer of myotubes. 
	NOTE: Omit wells on the edges of the plate because they are highly susceptible to evaporation. Vary the plate setup for experimental replicates to avoid systematic errors.</li>
	<li>Perform desired assay on differentiated myotubes within 1-2 days of full differentiation (Day 4-6 from start of differentiation). Recommended instruments and reagents for measurement of oxygen consumption rates are listed in the Table of Materials.</li>
</ol>

<ol>
	<li>Srikanthan, P., Karlamangla, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+mass+index+as+a+predictor+of+longevity+in+older+adults.">Muscle mass index as a predictor of longevity in older adults.</a> American Journal of Medicine. 127 (6), 547-553 (2014).</li><li>Lee-Young, R. S., Kang, L., Ayala, J. E., Wasserman, D. H., Fueger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological+regulation+of+glucose+flux+into+muscle+in+vivo.">The physiological regulation of glucose flux into muscle in vivo.</a> Journal of Experimental Biology. 214 (2), 254-262 (2010).</li><li>Sj&#248;berg, K. A., 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=Exercise+increases+human+skeletal+muscle+insulin+sensitivity+via+coordinated+increases+in+microvascular+perfusion+and+molecular+signaling.">Exercise increases human skeletal muscle insulin sensitivity via coordinated increases in microvascular perfusion and molecular signaling.</a> Diabetes. 66 (6), 1501-1510 (2017).</li><li>Girgis, C. M., Clifton-Bligh, R. J., Mokbel, N., Cheng, K., Gunton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D+signaling+regulates+proliferation,+differentiation,+and+myotube+size+in+C2C12+skeletal+muscle+cells.">Vitamin D signaling regulates proliferation, differentiation, and myotube size in C2C12 skeletal muscle cells.</a> Endocrinology. 155 (2), 347-357 (2014).</li><li>Smith, J., Merrick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+skeletal+muscle+microexplant+culture+and+isolation+of+skeletal+muscle+stem+cells.">Embryonic skeletal muscle microexplant culture and isolation of skeletal muscle stem cells.</a> Methods in Molecular Biology. 633, 29-56 (2010).</li><li>Mokbel, N., 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=K7del+is+a+common+TPM2+gene+mutation+associated+with+nemaline+myopathy+and+raised+myofibre+calcium+sensitivity.">K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.</a> Brain. 136 (2), 494-507 (2013).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270, 725-727 (1977).</li><li>Rando, T. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+myoblast+purification,+characterization,+and+transplantation+for+cell-mediated+gene+therapy.">Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.</a> Journal of Cell Biology. 125 (6), 1275-1287 (1994).</li><li>Musar&#242;, A., Carosio, S. <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+Satellite+Cells+from+Mouse+Skeletal+Muscle.">Isolation and Culture of Satellite Cells from Mouse Skeletal Muscle.</a> Methods in Molecular Biology. 1553, 155-167 (2017).</li><li>Smolina, N., Bruton, J., Kostareva, A., Sejersen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+mitochondrial+respiration+as+an+indicator+of+cellular+metabolism+and+fitness.">Assaying mitochondrial respiration as an indicator of cellular metabolism and fitness.</a> Methods in Molecular Biology. 1601, 79-87 (2017).</li><li>Elliott, B., Renshaw, D., Getting, S., Mackenzie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+role+of+myostatin+in+skeletal+muscle+and+whole+body+homeostasis.">The central role of myostatin in skeletal muscle and whole body homeostasis.</a> Acta Physiologica. 205 (3), 324-340 (2012).</li><li>Smolina, N., Kostareva, A., Bruton, J., Karpushev, A., Sjoberg, G., Sejersen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+murine+myotubes+as+a+model+for+investigating+muscular+dystrophy.">Primary murine myotubes as a model for investigating muscular dystrophy.</a> BioMed Research International. , (2015).</li><li>Nedachi, T., Fujita, H., Kanzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+C+2+C+12+myotube+model+for+studying+exercise-inducible+responses+in+skeletal+muscle.">Contractile C 2 C 12 myotube model for studying exercise-inducible responses in skeletal muscle.</a> American Journal of Physiology-Endocrinology and Metabolism. 295 (5), E1191-E1204 (2008).</li><li>Chen, M. B., 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=Impaired+activation+of+AMP-kinase+and+fatty+acid+oxidation+by+globular+adiponectin+in+cultured+human+skeletal+muscle+of+obese+type+2+diabetics.">Impaired activation of AMP-kinase and fatty acid oxidation by globular adiponectin in cultured human skeletal muscle of obese type 2 diabetics.</a> Journal of Clinical Endocrinology and Metabolism. 90 (6), 3665-3672 (2005).</li><li>Douillard-Guilloux, G., Mouly, V., Caillaud, C., Richard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+murine+muscle+cells+from+lysosomal+&#945;-glucosidase+deficient+mice:+A+new+tool+to+study+pathophysiology+and+assess+therapeutic+strategies+for+Pompe+disease.">Immortalization of murine muscle cells from lysosomal &#945;-glucosidase deficient mice: A new tool to study pathophysiology and assess therapeutic strategies for Pompe disease.</a> Biochemical and Biophysical Research Communications. 388 (2), 333-338 (2009).</li><li>Varga, B., 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=Myotube+elasticity+of+an+amyotrophic+lateral+sclerosis+mouse+model.">Myotube elasticity of an amyotrophic lateral sclerosis mouse model.</a> Scientific Reports. 8 (1), 5917 (2018).</li><li>Kriebs, A., 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=Circadian+repressors+CRY1+and+CRY2+broadly+interact+with+nuclear+receptors+and+modulate+transcriptional+activity.">Circadian repressors CRY1 and CRY2 broadly interact with nuclear receptors and modulate transcriptional activity.</a> Proceedings of the National Academy of Sciences. 114 (33), 8776-8781 (2017).</li><li>Cho, Y., 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=Perm1+enhances+mitochondrial+biogenesis,+oxidative+capacity,+and+fatigue+resistance+in+adult+skeletal+muscle.">Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.</a> FASEB Journal. 30 (2), 674-687 (2016).</li></ol>

Skeletal muscle is vital for the establishment and maintenance of metabolic homeostasis11. The study of muscle physiology is complicated by interindividual variability, as well as difficulty in obtaining samples, particularly in the case of human studies. Cultured primary myotubes have been shown to recapitulate many features of muscle physiology, including calcium homeostasis12, regeneration of damaged muscle tissue5, metabolic alterations in respon...

While often cited as an indication of overall metabolic health, multiple studies have shown that body mass index (BMI) in older adults is not consistently associated with higher risk of mortality. To date, the only factor shown to be consistent with reduced mortality in this population is increased muscle mass1. Muscle tissue represents one of the largest sources of insulin-sensitive cells in the body, and is therefore critical in the maintenance of overall metabolic homeostasis2. Activation of skeletal muscle tissue via exercise is associated with increases in both local insulin sensitivity and overall metabolic health3. While in vivo models are essential for studying muscle physiology and the impact of muscle function on integrated metabolism, primary cultures of myotubes provide a tractable system that reduces the complexity of animal studies.
Myoblasts derived from post-natal muscles can be used to study the impact of numerous treatment and growth conditions in a highly reproducible manner. This has long been recognized and several methods for myoblast isolation and culture have been described4,5,6,7,8,9. Some of these methods use neonatal muscles and yield relatively low numbers of myoblasts5,8, requiring several animals for larger scale studies. Also, most widely used methods for culturing myoblasts use &#34;pre-plating&#34; to enrich for myoblasts, which are less adherent than other cell types. We have found the alternative enrichment method described here to be much more efficient and reproducible for enriching a highly proliferative myoblast population. In summary, this protocol enables the isolation of highly proliferative myoblasts from young adult muscle explants, via outgrowth into culture media. Myoblasts can be harvested repeatedly, over several days, rapidly expanded, and induced to differentiate into myotubes. This protocol reproducibly generates a large number of healthy myoblast cells that robustly differentiate into spontaneously twitching myotubes. It has enabled us to study metabolism and circadian rhythms in primary myotubes of mice of a variety of genotypes. Finally, we include methods for preparing myotubes for the study of oxidative metabolism, using measurements of oxygen consumption rates in 96-well plates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Coating Solution:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Life Technologies</td>
			<td>A1064401</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Fisher</td>
			<td>CB40234A</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Plating Media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Life Technologies</td>
			<td>16000044</td>
			<td>20 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 &#176;C water bath for 20 minutes</td>
		</tr>
		<tr>
			<td>Amniomax</td>
			<td>Life Technologies</td>
			<td>12556023</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Myoblast Media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Life Technologies</td>
			<td>16000044</td>
			<td>10 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 &#176;C water bath for 20 minutes</td>
		</tr>
		<tr>
			<td>Amniomax</td>
			<td>Life Technologies</td>
			<td>12556023</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Differentiation Media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>Heat Inactivated Horse Serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Insulin-Selenium-Transferrin</td>
			<td>Life Technologies</td>
			<td>41400045</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Other Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>472301</td>
			<td>Prepare as 10% DMSO in Myoblast Media for freezing cells</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>VWR</td>
			<td>21427-648</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm plate</td>
			<td>VWR</td>
			<td>734-2318</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm plate</td>
			<td>VWR</td>
			<td>25382-428 (CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 Flasks</td>
			<td>ThermoFisher</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>ThermoFisher</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15mL)</td>
			<td>BioPioneer</td>
			<td>CNT-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Consumption Rates:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent</td>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Instrument used to measure oxygen consumption rates read out by acidification of the extracellular media</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent</td>
			<td>102416-100</td>
			<td>96-well plates for use in XFe96 Analyzer</td>
		</tr>
		<tr>
			<td>Seahorse XF Cell Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103015-100</td>
			<td>components may be purchased from other suppliers once assay is established; some recommendations are listed below</td>
		</tr>
		<tr>
			<td>Seahorse XF Palmitate-BSA FAO substrate</td>
			<td>Agilent</td>
			<td>102720-100</td>
			<td>components may be purchased from other suppliers once assay is established; some recommendations are listed below</td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>Sigma</td>
			<td>P5585-10G</td>
			<td>for measurement of fatty acid oxidation</td>
		</tr>
		<tr>
			<td>carnitine</td>
			<td>Sigma</td>
			<td>C0283-5G</td>
			<td>for measurement of fatty acid oxidation</td>
		</tr>
		<tr>
			<td>Etomoxir</td>
			<td>Sigma</td>
			<td>E1905</td>
			<td>for measurement of fatty acid oxidation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td>used as control or in conjugation with palmitic acid for use in measurement of fatty acid oxidation</td>
		</tr>
	</tbody>
</table>

isolation and differentiation of primary myoblasts from mouse skeletal muscle explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Following Section 1 of the provided protocol should yield primary cells emerging from the explants that will be visible under a standard light microscope (Figure 2). A heterogeneous cell population will be seen growing out of and surrounding each muscle tissue explant. Myoblasts will appear as small, round, bright spheres. Following Section 2 of the protocol will yield early harvests of myoblasts from tissue explants, which will contain few cells and will be ...

Watch this Scientific Journal Video about Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants at JoVE.com

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

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Developmental Biology

16.9K Views.  Department of Molecular Medicine. Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Video: Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...