Name: isolation of quiescent stem cell populations from individual skeletal muscles
Uploaded: 2022-12-09T13:45:00
Description: Watch this Scientific Journal Video about Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles at JoVE.com

Cell sorting was performed at the FACS Core Facility, Aarhus University, Denmark. Figures were created using Biorender.com. We thank Dr. J. Farup for sharing the rabbit anti-PDGFRa antibody. This work was supported by an AUFF Starting Grant to E.P. and Start Package grants from&#160;NovoNordiskFonden to E.P. (0071113) and to A.D.M. (0071116).

The present protocol was performed in accordance with animal care guidelines at Aarhus University and local ethics regulations.
NOTE: Make sure to comply with the regulations of the local ethical committee for animal experimentation and handling of post-mortem rodent samples. Mice are a potential source of allergens; if available, turn on exhaust ventilation and place it over the workspace to avoid excessive exposure to allergens. Alternatively, wear a face mask if the experiment is carried ou...

<ol>
	<li>Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cell+of+skeletal+muscle+fibers.">Satellite cell of skeletal muscle fibers.</a> The Journal of Biophysical and Biochemical Cytology. 9 (2), 493-495 (1961).</li><li>Relaix, F., 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=Perspectives+on+skeletal+muscle+stem+cells.">Perspectives on skeletal muscle stem cells.</a> Nature Communications. 12 (1), 692 (2021).</li><li>Cheung, T. H., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+stem+cell+quiescence.">Molecular regulation of stem cell quiescence.</a> Nature Reviews. Molecular Cell Biology. 14 (6), 329-340 (2013).</li><li>Kann, A. P., Hung, M., Krauss, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+contact+and+signaling+in+the+muscle+stem+cell+niche.">Cell-cell contact and signaling in the muscle stem cell niche.</a> Current Opinion in Cell Biology. 73, 78-83 (2021).</li><li>Tedesco, F. S., Dellavalle, A., Diaz-Manera, J., Messina, G., Cossu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repairing+skeletal+muscle:+regenerative+potential+of+skeletal+muscle+stem+cells.">Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells.</a> Journal of Clinical Investigation. 120 (1), 11-19 (2010).</li><li>Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A., Kardon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+connective+tissue+fibroblasts+and+their+interactions+are+crucial+for+muscle+regeneration.">Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration.</a> Development. 138 (17), 3625-3637 (2011).</li><li>Lepper, C., Partridge, T. A., Fan, C. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+absolute+requirement+for+Pax7-positive+satellite+cells+in+acute+injury-induced+skeletal+muscle+regeneration.">An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.</a> Development. 138 (17), 3639-3646 (2011).</li><li>Sambasivan, R., 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=Pax7-expressing+satellite+cells+are+indispensable+for+adult+skeletal+muscle+regeneration.">Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.</a> Development. 138 (17), 3647-3656 (2011).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., 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=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. 456 (7221), 502-506 (2008).</li><li>Joe, A. W. 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=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nature Cell Biology. 12 (2), 153-163 (2010).</li><li>Wosczyna, M. 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=Mesenchymal+stromal+cells+are+required+for+regeneration+and+homeostatic+maintenance+of+skeletal+muscle.">Mesenchymal stromal cells are required for regeneration and homeostatic maintenance of skeletal muscle.</a> Cell Reports. 27 (7), 2029-2035 (2019).</li><li>Uezumi, A., Fukada, S. -. I., Yamamoto, N., Takeda, S., Tsuchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+progenitors+distinct+from+satellite+cells+contribute+to+ectopic+fat+cell+formation+in+skeletal+muscle.">Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.</a> Nature Cell Biology. 12 (2), 143-152 (2010).</li><li>Seale, P., Sabourin, L. A., Girgis-Gabardo, A., Mansouri, A., Gruss, P., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7+Is+Required+for+the+Specification+of+Myogenic+Satellite+Cells.">Pax7 Is Required for the Specification of Myogenic Satellite Cells.</a> Cell. 102 (6), 777-786 (2000).</li><li>Shea, K. L., 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=Sprouty1+regulates+reversible+quiescence+of+a+self-renewing+adult+muscle+stem+cell+pool+during+regeneration.">Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration.</a> Cell Stem Cell. 6 (2), 117-129 (2010).</li><li>Fukada, S. -. I., 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=Molecular+signature+of+quiescent+satellite+cells+in+adult+skeletal+muscle.">Molecular signature of quiescent satellite cells in adult skeletal muscle.</a> Stem Cells. 25 (10), 2448-2459 (2007).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nature Protocols. 10 (10), 1612-1624 (2015).</li><li>Joe, A., Wang, J., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+adipogenic+progenitors+from+skeletal+muscle.">Prospective isolation of adipogenic progenitors from skeletal muscle.</a> Journal of Investigative Medicine. 55 (1), 124 (2007).</li><li>Yi, L., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+progenitors+from+skeletal+muscle.">Purification of progenitors from skeletal muscle.</a> Journal of Visualized Experiments. (49), e2476 (2011).</li><li>Sherwood, R. I., 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=Isolation+of+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119 (4), 543-554 (2004).</li><li>Montarras, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309 (5743), 2064-2067 (2005).</li><li>Conboy, M. J., Cerletti, M., Wagers, A. J., Conboy, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-analysis+and+FACS+sorting+of+adult+muscle+fiber-associated+stem/precursor+cells.">Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells.</a> Methods In Molecular Biology. 621, 165-173 (2010).</li><li>de Morree, 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=Alternative+polyadenylation+of+Pax3+controls+muscle+stem+cell+fate+and+muscle+function.">Alternative polyadenylation of Pax3 controls muscle stem cell fate and muscle function.</a> Science. 366 (6466), 734-738 (2019).</li><li>Stuelsatz, P., 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=Extraocular+muscle+satellite+cells+are+high+performance+myo-engines+retaining+efficient+regenerative+capacity+in+dystrophin+deficiency.">Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency.</a> Developmental Biology. 397 (1), 31-44 (2015).</li><li>Mookhtiar, K., Randall Steinbrink, D., Van Wart, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+hydrolysis+of+collagen-like+peptides+by+class+I+and+class+II+Clostridium+histolyticum+collagenases:+evidence+for+both+endopeptidase+and+tripeptidylcarboxypeptidase+activities.">Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities.</a> Biochemistry. 24 (23), 6527-6533 (1985).</li><li>Stenn, K. S., Link, R., Moellmann, G., Madri, J., Kuklinska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispase,+a+neutral+protease+from+Bacillus+polymyxa,+is+a+powerful+fibronectinase+and+type+IV+collagenase.">Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase.</a> The Journal of Investigative Dermatology. 93 (2), 287-290 (1989).</li><li>Baghdadi, 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=Reciprocal+signalling+by+Notch-Collagen+V-CALCR+retains+muscle+stem+cells+in+their+niche.">Reciprocal signalling by Notch-Collagen V-CALCR retains muscle stem cells in their niche.</a> Nature. 557 (7707), 714-718 (2018).</li><li>van Velthoven, C. T. J., de Morree, A., Egner, I. M., Brett, J. O., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+quiescent+muscle+stem+cells+in+vivo.">Transcriptional profiling of quiescent muscle stem cells in vivo.</a> Cell Reports. 21 (7), 1994-2004 (2017).</li><li>Machado, L., 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=In+situ+fixation+redefines+quiescence+and+early+activation+of+skeletal+muscle+stem+cells.">In situ fixation redefines quiescence and early activation of skeletal muscle stem cells.</a> Cell Reports. 21 (7), 1982-1993 (2017).</li><li>Machado, L., 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=Tissue+damage+induces+a+conserved+stress+response+that+initiates+quiescent+muscle+stem+cell+activation.">Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation.</a> Cell Stem Cell. 28 (6), 1125-1135 (2021).</li><li>vanden Brink, S. C., 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=Single-cell+sequencing+reveals+dissociation-induced+gene+expression+in+tissue+subpopulations.">Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations.</a> Nature Methods. 14 (10), 935-936 (2017).</li><li>Moore, D. K., Motaung, B., du Plessis, N., Shabangu, A. N., Loxton, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SU-IRG+consortium+isolation+of+B-cells+using+Miltenyi+MACS+bead+isolation+kits.">SU-IRG consortium isolation of B-cells using Miltenyi MACS bead isolation kits.</a> PloS One. 14 (3), 0213832 (2019).</li><li>Liou, Y. -. R., Wang, Y. -. H., Lee, C. -. Y., Li, P. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buoyancy-activated+cell+sorting+using+targeted+biotinylated+albumin+microbubbles.">Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.</a> PloS One. 10 (5), 0125036 (2015).</li><li>Brett, J. O., 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+rejuvenates+quiescent+skeletal+muscle+stem+cells+in+old+mice+through+restoration+of+Cyclin+D1.">Exercise rejuvenates quiescent skeletal muscle stem cells in old mice through restoration of Cyclin D1.</a> Nature Metabolism. 2 (4), 307-317 (2020).</li><li>Tabula Muris Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+transcriptomic+atlas+characterizes+ageing+tissues+in+the+mouse.">A single-cell transcriptomic atlas characterizes ageing tissues in the mouse.</a> Nature. 583 (7817), 590-595 (2020).</li><li>de Morr&#233;e, 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=Staufen1+inhibits+MyoD+translation+to+actively+maintain+muscle+stem+cell+quiescence.">Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence.</a> Proceedings of the National Academy of Sciences. 114 (43), 8996-9005 (2017).</li></ol>

Several steps are key in the execution of this protocol to achieve good yields. The individual muscles have a small volume compared to the amount of muscle used in bulk isolation protocols. This results in a risk of the muscle drying out during the dissection, which reduces yield. To prevent this, it is important to add medium to the muscles immediately after dissection. In addition, if dissection is taking longer, the skin can be removed from one limb at a time to reduce the time of exposure of the muscles to air. The s...

Skeletal muscle has a high capacity for regeneration due to the presence of muscle stem cells (MuSCs). MuSCs are located on the myofibers, underneath the basal lamina, and reside in a quiescent state of prolonged, reversible cell cycle exit1,2,3,4. Upon injury, MuSCs activate and enter the cell cycle to give rise to amplifying progenitors that can differentiate and fuse to form new myofibers2,5. Previous work has showed that MuSCs are absolutely essential for muscle regeneration6,7,8. Moreover, a single MuSC can engraft and generate both new stem cells and new myofibers9. Skeletal muscle also harbors a population of mesenchymal stromal cells called fibro-adipogenic progenitors (FAPs), which play a crucial role in supporting MuSC function during muscle regeneration6,10,11,12.
Due to their potential to coordinate muscle regeneration, there has been tremendous interest in understanding how MuSCs and FAPs work. Quiescent MuSCs are marked by expression of the transcription factors Pax7 and Sprouty1, and the cell surface protein calcitonin receptor, whereas quiescent FAPs are marked by the cell surface protein platelet-derived growth factor receptor alpha (PDGFRa)10,12,13,14,15. Previous studies have showed that MuSCs and FAPs could be purified from skeletal muscles using cell surface markers and fluorescence-activated cell sorting (FACS)9,15,16,17,18,19,20,21. While these protocols have greatly advanced the ability to study MuSCs and FAPs, one drawback is that most of these protocols require the isolation of MuSCs from a pool of different muscle tissues. Recent work from us and others have revealed differences in cell phenotype and gene expression levels between MuSCs isolated from different tissues22,23. MuSCs from the diaphragm, triceps, and gracilis show faster activation than MuSCs from lower hindlimb muscles22, while MuSCs from extraocular muscle show faster differentiation than MuSCs from the diaphragm and lower hindlimb muscles23.
This protocol describes the isolation of MuSCs and FAPs from individual skeletal muscles (Figure 1). This includes the dissection of the diaphragm, triceps, gracilis, tibialis anterior (TA), soleus, extensor digitorum longus (EDL), gastrocnemius (GA), and masseter muscles. Dissected muscles are subsequently dissociated by enzymatic digestion using collagenase II (a protease that specifically targets the Pro-X-Gly-Pro amino sequence in collagen, enabling the degradation of connective tissue and tissue dissociation24) and dispase (a protease that cleaves fibronectin and collagen IV, enabling further cell dissociation25). MuSCs and FAPs are isolated from single-cell suspensions by FACS. As examples of downstream assays for cell analysis, stem cell activation is determined by assaying 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation, while cell purity is determined by immunofluorescence staining for the cell-type specific markers Pax7 and PDGFRa.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL tube( PCR performance tested, PP, 30,000 xg, DNA/DNase-/RNase-free, Low DNA binding, Sterile )</td>
			
			<td>Sarstedt AG &#38; Co. KG, Hounisen Laboratorieudstyr A/S</td>
<td>72.706.700</td>			
<td>1.5 mL tube</td>
		</tr>
		<tr>
			<td>15 mL tube (PP/HD-PE, 20,000 xg, IVD/CE, IATA, DNA/DNase-/RNase-free, Non-cytotoxic, pyrogen free, Sterile)</td>
			
			<td>Sarstedt AG &#38; Co. KG, Hounisen Laboratorieudstyr A/S</td>
<td>62.554.502</td>			
<td>15 mL tube</td>
		</tr>
		<tr>
			<td>5 mL polystyrene round-bottom tube</td>
			
			<td>Falcon, Fisher Scientific&#160;</td>
<td>352054</td>			
<td>FACS tube without strainer cap</td>
		</tr>
		<tr>
			<td>5 mL polystyrene Round-bottom tube with cell-strainer cap</td>
			
			<td>Falcon, Fisher Scientific&#160;&#160;</td>
<td>352235</td>			
<td>FACS tube with strainer cap</td>
		</tr>
		<tr>
			<td>5 mL tube (PP, non sterile autoclavable)</td>
			
			<td>VWR collection</td>
<td>525.0946</td>			
<td>5 mL tube</td>
		</tr>
		<tr>
			<td>50 mL tube( PP/HD-PE, 20,000 xg, IVD/CE, ADR, DNA/DNase-/RNase-free, non-cytotoxic, pyrogen free, Sterile)</td>
			
			<td>Sarstedt AG &#38; Co. KG, Hounisen Laboratorieudstyr A/S</td>
<td>62.547.254</td>			
<td>50 mL tube</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Donkey anti-rabbit IgG (H+L)</td>
			
			<td>Invitrogen, Thermo Fisher</td>
<td>Lot: 2387458 (Cat # A31572)</td>			
<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey-anti mouse IgG (H+L)</td>
			
			<td>Invitrogen, Thermo Fisher</td>
<td>Lot: 2420713 (Cat#A31571)</td>			
<td></td>
		</tr>
		<tr>
			<td>ARIA 3</td>
			
			<td>BD</td>
<td></td>			
<td>FACS, Core facility Aarhus University</td>
		</tr>
		<tr>
			<td>Centrifuge 5810</td>
			
			<td>eppendorf</td>
<td>EP022628188</td>			
<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye</td>
			
			<td>Invitrogen, Thermo Fisher</td>
<td>Lot: 2387287 (Cat# C10337)</td>			
<td>Cell Proliferation Kit</td>
		</tr>
		<tr>
			<td>Collagen from calf-skin&#160;</td>
			
			<td>Bioreagent, Sigma Aldrich&#160;</td>
<td>Source: SLCK6209 (Cat# C8919)</td>			
<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			
			<td>Worthington, Fisher Scientific&#160;</td>
<td>Lot: 40H20248 (cat# L5004177 )</td>			
<td>Collagenase</td>
		</tr>
		<tr>
			<td>Dispase</td>
			
			<td>Gibco, Fisher Scientific&#160;</td>
<td>Lot: 2309415 (cat# 17105-041 )</td>			
<td>Dispase</td>
		</tr>
		<tr>
			<td>Donkey serum (non-sterile)</td>
			
			<td>Sigma Aldrich, Merck</td>
<td>Lot: 2826455 (Cat# S30-100mL)</td>			
<td></td>
		</tr>
		<tr>
			<td>Dumont nr. 5, 110 mm</td>
			
			<td>Dumont, Hounisen Laboratorieudstyr A/S</td>
<td>1606.327</td>			
<td>Straight forceps with fine tips</td>
		</tr>
		<tr>
			<td>Dumont nr. 7, 115 mm</td>
			
			<td>Dumont, Hounisen Laboratorieudstyr A/S</td>
<td>1606.335</td>			
<td>Curved forceps</td>
		</tr>
		<tr>
			<td>F-10 Nutrient mixture (Ham) (1x), +L-glutamine</td>
			
			<td>Gibco, Fisher Scientific&#160;</td>
<td>Lot. 2453614 (cat# 31550-023)</td>			
<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD31</td>
			
			<td>BioLegend, NordicBioSite</td>
<td>MEC13.3 (Cat # 102506)</td>			
<td></td>
		</tr>
		<tr>
			<td>FITC Anti-mouse CD45</td>
			
			<td>BioLegend, NordicBioSite</td>
<td>30-F11 (Cat# 103108)</td>			
<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid (100%)</td>
			
			<td>EMSURE, Merck&#160;&#160;</td>
<td>K44104563 9Cat # 1000631000)</td>			
<td></td>
		</tr>
		<tr>
			<td>Head over head mini-tube rotator&#160;</td>
			
			<td>Fisher Scientific&#160;</td>
<td>15534080 (Model no. 88861052)</td>			
<td>Head over head mini-tube rotator</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			
			<td>Gibco, Fisher Scientific&#160;</td>
<td>Lot. 2482639 (cat# 10368902 )</td>			
<td></td>
		</tr>
		<tr>
			<td>Isotemp SWB 15</td>
			
			<td>FisherBrand, Fisher Scientific</td>
<td>15325887</td>			
<td>Shaking water bath</td>
		</tr>
		<tr>
			<td>MS2 mini-shaker&#160;</td>
			
			<td>IKA&#160;</td>
<td></td>			
<td>Vortex unit</td>
		</tr>
		<tr>
			<td>Needle 20 G (0.9 mm x 25 mm)</td>
			
			<td>BD microlance, Fisher Scientific&#160;</td>
<td>304827</td>			
<td>20G needle&#160;</td>
		</tr>
		<tr>
			<td>Neutral formalin buffer 10%</td>
			
			<td>CellPath, Hounisen Laboratorieudstyr A/S</td>
<td>Lot: 03822014 (Cat # HOU/1000.1002)</td>			
<td></td>
		</tr>
		<tr>
			<td>Non-pyrogenic cell strainer (40 &#181;M)</td>
			
			<td>Sarstedt AG &#38; Co. KG, Hounisen Laboratorieudstyr A/S</td>
<td>83.3945.040</td>			
<td>Cell strainer&#160;</td>
		</tr>
		<tr>
			<td>Pacific Blue anti-mouse Ly-6A/E (Sca-1)</td>
			
			<td>BioLegend, NordicBioSite</td>
<td>D7 (Cat# 108120)</td>			
<td></td>
		</tr>
		<tr>
			<td>Pax7 primary antibody</td>
			
			<td>DSHB</td>
<td>Lot: 2/3/22-282ug/mL (Cat# AB 528428)</td>			
<td></td>
		</tr>
		<tr>
			<td>PBS 10x powder concentrate</td>
			
			<td>Fisher BioReagents, Fisher Scientific</td>
<td>BP665-1</td>			
<td></td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-mouse CD106 (VCAM1)</td>
			
			<td>BioLegend, NordicBioSite</td>
<td>429 (MVCAM.A) (Cat # 105720)</td>			
<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			
			<td>Gibco, Fisher Scientific&#160;</td>
<td>Lot. 163589 (cat# 11548876 )</td>			
<td></td>
		</tr>
		<tr>
			<td>Pipette tips p10</td>
			
			<td>Art tips, self sealing barrier, Thermo Scientific</td>
<td>2140-05</td>			
<td>Low retention, pre-sterilized, filter tips</td>
		</tr>
		<tr>
			<td>Pipette tips p1000</td>
			
			<td>Art tips, self sealing barrier, Thermo Scientific</td>
<td>2279-05</td>			
<td>Low retention, pre-sterilized, filter tips</td>
		</tr>
		<tr>
			<td>Pipette tips p20</td>
			
			<td>Art tips, self sealing barrier, Thermo Scientific</td>
<td>2149P-05</td>			
<td>Low retention, pre-sterilized, filter tips</td>
		</tr>
		<tr>
			<td>Pipette tips p200</td>
			
			<td>Art tips, self sealing barrier, Thermo Scientific</td>
<td>2069-05</td>			
<td>Low retention, pre-sterilized, filter tips</td>
		</tr>
		<tr>
			<td>Protective underpad</td>
			
			<td>Abena&#160;</td>
<td>ACTC-7712</td>			
<td>&#160;60 x 40cm, 8 layers</td>
		</tr>
		<tr>
			<td>Rainin, pipet-lite XLS</td>
			
			<td>Mettler Toledo, Thermo Scientific&#160;</td>
<td>2140-05, 2149P-05, 2279-05, 2069-05</td>			
<td>Pipettes (P10, P20, P200, P1000)</td>
		</tr>
		<tr>
			<td>Recombinant anti-PDGFR-alpha</td>
			
			<td>RabMAb, abcam</td>
<td>AB134123</td>			
<td></td>
		</tr>
		<tr>
			<td>Scalpel (shaft no. 3)</td>
			
			<td>Hounisen, Hounisen Laboratorieudstyr A/S</td>
<td>1902.502</td>			
<td>Scalpel</td>
		</tr>
		<tr>
			<td>Scalpel blade no. 11</td>
			
			<td>Heinz Herenz, Hounisen Laboratorieudstyr A/S</td>
<td>1902.0911</td>			
<td>Scalpel</td>
		</tr>
		<tr>
			<td>Scanlaf mars</td>
			
			<td>Labogene</td>
<td>class 2 cabinet: Mars</td>			
<td>Flow bench</td>
		</tr>
		<tr>
			<td>ScanR</td>
			
			<td>Olympus</td>
<td></td>			
<td>Microscope, Core facility Aarhus University</td>
		</tr>
		<tr>
			<td>Scissors</td>
			
			<td>FST</td>
<td>14568-09</td>			
<td></td>
		</tr>
		<tr>
			<td>Series 8000 DH</td>
			
			<td>Thermo Scientific</td>
<td>3540-MAR</td>			
<td>Incubator</td>
		</tr>
		<tr>
			<td>Serological pipette 10 mL</td>
			
			<td>VWR</td>
<td>612-3700</td>			
<td>Sterile, non-pyrogenic</td>
		</tr>
		<tr>
			<td>Serological pipette 5 mL</td>
			
			<td>VWR, Avantor delivered by VWR</td>
<td>612-3702</td>			
<td>Sterile, non-pyrogenic</td>
		</tr>
		<tr>
			<td>Syringe 5 mL, Luer tip (6%), sterile&#160;</td>
			
			<td>BD Emerald, Fisher Scientific</td>
<td>307731</td>			
<td>Syringe</td>
		</tr>
		<tr>
			<td>TC Dish 100, standard</td>
			
			<td>Sarstedt AG &#38; Co. KG, Hounisen Laboratorieudstyr A/S</td>
<td>83.3902</td>			
<td>Petri dish&#160;</td>
		</tr>
		<tr>
			<td>Tissue Culture (TC)-treated surface, black polystyrene, flat bottom, sterile, lid, pack of 20</td>
			
			<td>Corning, Sigma Aldrich</td>
<td>3764</td>			
<td>96-well Half bottom plate</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			
			<td>Sigma Aldrich, Merck</td>
<td>Source: SLCJ6163 (Cat # T8787)</td>			
<td></td>
		</tr>
	</tbody>
</table>

isolation of quiescent stem cell populations from individual skeletal muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

Following the protocol for individual skeletal muscle isolation (Figure 2), the gracilis, TA, EDL, GA, soleus, triceps, masseter, and diaphragm muscles were isolated from three Swiss male outbred mice that had been discontinued from a local breeding program (Figure 2). Following tissue dissociation and antibody staining, MuSCs and FAPs from the individual muscles were purified by FACS (Figure 3). The initial gating was obtained with...

Watch this Scientific Journal Video about Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles at JoVE.com

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...

Research

JoVE Journal

Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

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

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

Mitochondrial Isolation from Skeletal Muscle

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

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

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

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

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

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

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...