This work was supported by NIAMS AR064873, Epigen Project PB. P01.001.019/Progetto Bandiera Epigenomica IFT to L.L.

Mice were bred and maintained according to the standard animal facility procedures, and all experimental protocols were approved by the Animal Welfare Assurance and the internal Animal Research Ethical Committee according to the Italian Ministry of Health and complied with the NIH Guide for the Care and Use of Laboratory Animals.
1. Muscle Injury and the In Vivo Block of Autophagic Flux
<ol>
	<li>Muscle injury.
	<ol>
		<li>To induce acute skeletal muscle injury, inject 20 &#181;L of 10 &#181;M cardiotoxin (CTX) stock directly in the left tibialis anterior (TA) muscle of 2 month-old C57BL/6J mice that weigh approximately 20 g. Use an equal number of males and females. Use the unperturbed contralateral limb as a control. 
		NOTE: 10 &#181;M CTX in 0.9% sodium chloride solution should be filtered (0.22 &#181;m PVDF filter) and stored at -20 &#176;C. Avoid repeated freeze-thaw cycles.</li>
		<li>Using an insulin syringe with a 30 G needle, inject CTX in the middle of the TA. To induce an accurate injury in the TA, ensure that the needle of the syringe enters near the distal tendon, 5 mm deep, from the bottom to the top of the muscle (Figure 1A).</li>
	</ol>
	</li>
	<li>Blockade of the autophagic flux in unperturbed and injured mice .
	<ol>
		<li>To assess the autophagic flux, 24 h Post-Injury (p.i.), administer 50 mg/kg chloroquine (CLQ) every 24 h for 4 d by intraperitoneal (IP) injection (Figure 1B).
		<ol>
			<li>Dissolve CLQ (10 mg/mL) in PBS 1X and filter it through a 0.2 &#181;m membrane. Weigh every mouse and calculate the amount of CLQ to inject. 
			NOTE: Prepare and use CLQ solution on the same day. CLQ stock can be stored for up to one month at -20 &#176;C. Since autophagy is a metabolic-related process, always perform CLQ treatment always at the same time (i.e.&#160;in the morning before 10 AM).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Muscle Tissue Sections
<ol>
	<li>Mouse sacrifice.
	<ol>
		<li>Euthanize the mice 5 days after the injury.</li>
		<li>Perform euthanasia by cervical dislocation or by CO2 (to be followed by cervical dislocation for confirmation). Since the autophagy process is related to mouse sleeping/eating habits, always sacrifice the mice at the same time, preferably in the morning (i.e.&#160;before 10 AM).</li>
	</ol>
	</li>
	<li>TA isolation and inclusion.
	<ol>
		<li>Before the dissection, spray the mouse with 70% ethanol.</li>
		<li>Using scissors make a small, perpendicular incision (3 mm long) on the dorsal skin of the mouse at the level of the hips. Cut the tail and the feet to simplify the skin removal. Pull the skin towards the tail and remove it to expose the underlying muscles; the TA muscle is easy to localize, as shown in Figure 2A.</li>
		<li>With the help of two forceps, grasp the distal tendons.</li>
		<li>Cut the distal tendons; the TA and extensor digitorium longus (EDL) tendons are often cut jointly and later separated (see step 2.2.6).</li>
		<li>Using forceps, hold the TA muscle by the tendon and carefully pull the muscle up toward the proximal end (near the knee; Figure 2B). 
		NOTE: At this point, the EDL and TA muscles are readily recognizable, the TA being larger and more superficial than the EDL.</li>
		<li>Separate the TA from the EDL muscle by pulling the two distal tendons in the opposite directions (Figure 2C).</li>
		<li>Cut the proximal tendon.</li>
		<li>Using forceps, remove the thin fascia that covers the muscle, without damaging the tissue.</li>
		<li>Remove excessive moisture in the sample with the help of a paper towel. Ensure that the muscle is neither wet, nor excessively dry, as excessive moisture will produce significant damage to the muscle and jeopardize the next staining.</li>
		<li>Place Optimal Cutting Temperature (OCT) compound at the bottom of a mold and place the muscle into it, in the orientation shown in Figure 2D. Add 100% isopentane to a beaker until it reaches a depth of approximately 3-4 cm. Place the beaker in contact with liquid nitrogen in a polystyrene box.</li>
		<li>Do not let the liquid nitrogen enter the beaker, as it will produce a bubbling foam, which can affect the inclusion and damage the muscle sections.</li>
		<li>Observe the isopentane and wait until it reaches the proper temperature (between -140 &#176;C and -150 &#176;C); at the appropriate temperature, a solid white layer of frozen isopentane will crystallize on the bottom of the beaker.</li>
		<li>If the isopentane entirely freezes solid, melt and chill it to freezing temperature before proceeding to the next step.</li>
		<li>Using prechilled forceps, delicately lower the mold into the isopentane for approximately 20-30 s. Place the frozen sample into a container with dry ice until it is transferred to a -80 &#176;C freezer. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Muscle tissue cryosections.
	<ol>
		<li>After at least one night at -80 &#176;C, perform cryosectioning.</li>
		<li>Take the cryostat knife and the anti-roll plate out of the -20 &#176;C freezer and place them on the respective supports into the cryostat cabinet.</li>
		<li>Put a drop of OCT on the sample stub and place on it the frozen sample, in vertical N-S orientation, as visualized in Figure 2D.</li>
		<li>Place the sample stub on the chuck.</li>
		<li>Without pulling the anti-roll plate down on the knife, make some cuts at 40 &#181;m to get rid of OCT that does not include muscle. When the muscle becomes visible, reduce the slice thickness to 7-8 &#181;m.</li>
		<li>Pull down the anti-roll plate until it is aligned with the edge of the knife or a little above it.</li>
		<li>Cut transversal sections 7-8 &#181;m thick and mount 3-4 slices per histological slide. 
		NOTE: The suggested cryostat temperature is between -15 &#176;C and -23 &#176;C. Cross-sections of the samples are needed for the subsequent analysis to pinpoint the muscle stem cells in the satellite niche position.</li>
		<li>To maximize the adherence of the section, keep the slides at room temperature for 10-15 min so to prevent their detachment during antibody incubation. 
		NOTE: The air-drying step could affect immunostaining, providing ambiguous results. Slides can be stored unfixed for several months at -80 &#176;C. The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Checking for muscle damage in cryosections of TA muscles by performing Hematoxylin Eosin (H&#38;E) staining.
	<ol>
		<li>To evaluate the quality of the muscle (i.e.&#160;the effectiveness of the injury, muscle isolation and inclusion, and cryosections), perform an H&#38;E staining. For every experimental time point, fix one slide with 4% PFA for 10 min. After fixation, wash the slides well in several changes of 1x PBS. 
		NOTE: Caution. PFA is carcinogen and must be handled carefully.</li>
		<li>Take one slide for every experimental point and put them in a staining jar.</li>
		<li>Fill the jar with 9 g/L hematoxylin until the sections are covered and incubate for 8 min.</li>
		<li>Recycle the hematoxylin.</li>
		<li>Leave the jar under running water for 10 min to remove the excess of hematoxylin. 
		NOTE: Make sure not to flow the water directly onto the sections.</li>
		<li>Wash using sterilized water.</li>
		<li>Incubate the sections with eosin 0.5% (w/v) in acidified 90% ethanol for 1 min.</li>
		<li>Recycle the eosin.</li>
		<li>Wash twice with sterile water for 3 min/wash. 
		NOTE: Perform the following steps at a chemical hood.</li>
		<li>Wash the sections with 70% ethanol.</li>
		<li>Wash the sections with 90% ethanol.</li>
		<li>Wash the sections with 100 % ethanol.</li>
		<li>Incubate the sections with 0.879 g/mL o-Xylene. 
		NOTE: Caution. o-Xylene is flammable and moderately toxic. Manipulate it under chemical hood, wearing protective equipment, including gloves, a lab coat, and a mask.</li>
		<li>Recycle the o-Xylene.</li>
		<li>Put the slides over a piece of paper to dry.</li>
		<li>Close the slides using a quick-hardening, xylene-based mounting medium.</li>
		<li>Check the quality of the sections under an optical microscope at 10X magnification (see Figure 3) 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
3. Immunostaining for LC3 and MyoD in Injured Muscle Tissue Sections
<ol>
	<li>Fixation of muscle tissue sections.
	<ol>
		<li>Prepare an incubation chamber by wetting a piece of paper and placing it on the bottom of a closable plastic box to maintain a high level of humidity inside the chamber. Fix tissue sections with 4% paraformaldehyde (PFA) in 1x PBS for 10 min. 
		NOTE: A wet piece of paper is used to avoid drying the sample. Prepare fresh PFA or use PFA stored at -20 &#176;C. Caution. PFA is toxic; the powder must be handled with care when making the stock solution. This operation must be performed at a chemical hood, with protective equipment, including gloves, a lab coat, and a mask. Also, when in solution, the PFA should be manipulated carefully.</li>
		<li>Remove the PFA and wash the sections with 1x PBS for 5-7 min; repeat this step 3 times.</li>
	</ol>
	</li>
	<li>Permeabilization of muscle tissue sections.
	<ol>
		<li>Draw a hydrophobic barrier around the tissue sections using a pap pen. 
		NOTE: This step defines the surface around the tissue sections and minimizes the volume of antibodies described in steps 3.4, 3.6, 3.7, and 3.9.</li>
		<li>Cover the sections with 200 &#181;L of cold (-20 &#176;C) methanol and put the incubation chamber horizontally inside a freezer at -20 &#176;C for 5 min .</li>
		<li>Remove the incubation chamber from the freezer, aspirate the methanol, and wash the sections with1X PBS for 5-7 min at RT; repeat this step 3x.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Prepare a fresh block solution of 4% Bovine Serum Albumin&#160;(BSA) in PBS 1x. 
		NOTE: BSA solution can be stored up for 1 week at 4 &#176;C.</li>
		<li>Incubate the sections with block solution for at least 60 min at RT.</li>
		<li>Remove the blocking solution.</li>
		<li>Prepare 100 &#181;L of anti-Fab mix by diluting 20 &#181;g/mL anti-Fab in 1x PBS. Incubate the sections with an unconjugated affinity-purified F(ab) fragment of anti-mouse IgG for 1 h at RT.</li>
	</ol>
	</li>
	<li>Primary antibody incubation.
	<ol>
		<li>Prepare 100 &#181;L of primary antibody mix for each slide by dissolving LC3 and MyoD antibodies in 4% BSA in 1x PBS. Use 5 &#181;g/mL of anti-LC3 (rabbit polyclonal antibody) and 10 mg/L of anti-MyoD (mouse monoclonal antibody). Incubate the primary antibody mix overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Washes.
	<ol>
		<li>Remove the primary antibody mix.</li>
		<li>Wash the sections with 1% BSA in 1X PBS for 10 min; repeat this step 3x.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation.
	<ol>
		<li>Prepare 100 &#181;L of secondary antibody mix for each slide. Dissolve goat anti-rabbit 488 (5 &#181;g/mL) and goat anti-mouse 594 (5 &#181;g/mL) in 4% BSA in 1x PBS. 
		NOTE: Avoid excessive exposure to light to prevent fluorochrome bleaching. Perform the following steps in the dark.</li>
		<li>Incubate the sections with the secondary antibody mix for 45 min at RT.</li>
		<li>Remove the secondary antibody mix and wash the sections with 1x PBS for 5-7 min; repeat this step 3x.</li>
	</ol>
	</li>
	<li>Primary antibody incubation.
	<ol>
		<li>Dilute laminin-2 (&#945;-2-chain) monoclonal antibody (0.33 &#181;g/mL) in 4% BSA in 1x PBS using 100 &#181;L of mixture for each slide. Incubate the sections in the primary antibody mix for 1-2 h at RT.</li>
	</ol>
	</li>
	<li>Washes.
	<ol>
		<li>Remove the primary antibody mix.</li>
		<li>Wash the sections with 1% BSA in 1X PBS for 10 min; repeat this step 3x.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation.
	<ol>
		<li>Prepare 100 &#181;L of secondary antibody mix for each slide by diluting goat anti-rat 647 (5 &#181;g/mL) in 4% BSA in 1X PBS. Incubate the sections in secondary antibody mix for 45 min at RT.</li>
		<li>Remove the secondary antibody mix and wash the sections with 1x PBS for 5-7 min; repeat this step 3x.</li>
	</ol>
	</li>
	<li>4&#39;,6-Diamidino-2-phenylindole (DAPI) incubation.
	<ol>
		<li>Add 200 &#181;L of 300 nM DAPI solution in 1x PBS to each slide. Incubate for 5 min at RT. Store the DAPI solution at 4 &#176;C in the dark.</li>
		<li>Wash the sections with 1x PBS for 5-7 min; repeat this step 3x.</li>
	</ol>
	</li>
	<li>Mounting stained muscle sections.
	<ol>
		<li>Remove the excess of washing solution from the sections.</li>
		<li>Place 10 &#181;L of glycerol in 1x PBS (3:1) on the stained sections in the middle of the slide and add a coverslip, avoiding the formation of air bubbles.</li>
	</ol>
	</li>
</ol>
4. Confocal Microscopy Acquisition
<ol>
	<li>Acquire the images using a four-laser confocal microscope integrated with an image capture system and analytical software. 
	NOTE: MyoD is conjugated with a 594 dye that is a bright red-fluorescent dye, with excitation ideally suited to the 594 nm laser (excitation: 590 nm, emission: 617 nm). LC3 is conjugated with a 488 dye that is a bright green-fluorescent dye, with excitation ideally suited to the 488 nm laser line (excitation: 495 nm, emission: 519 nm). Laminin is conjugated with a 647 dye that is a bright far-red-fluorescent dye, with excitation ideally suited to the 647 nm laser line (excitation: 650 nm, emission: 668 nm).</li>
	<li>Place the slides in the microscope tray and use 63X magnification to detect nuclear signals.Manually focus on the regenerative areas by centering the fields of active regeneration, relying on muscle morphology (see Figure 4). 
	NOTE: While in unperturbed muscle, myofiber size is virtually constant (Figure 4A), injury-mediated muscle regeneration can be recognized by the appearance of smaller myofibers, which are the newly formed fibers after regeneration. Moreover, the muscle in active regeneration is characterized by an extensive infiltrate that is made of macrophages, fibro-adipogenic precursors, and cells that are localized to the damaged muscles to orchestrate muscle regeneration (Figure 4B).</li>
	<li>Evaluate the immunofluorescent staining in regenerative areas by focusing on MuSCs that are located under the basal lamina of the myofibers.</li>
	<li>Focus on MuSCs that are positive for MyoD staining and positioned within myofibers marked by laminin staining (see Figure 4B, white arrows).</li>
	<li>Use at least 3 mice/experimental group and use the microscope to acquire 63X magnification images of at least 7 fields for each experimental group.</li>
	<li>Once the regenerative areas are identified, focus using the DAPI staining and then adjust the other single channels.
	<ol>
		<li>Use the following microscope settings: Channel A594 Pinhole 1.2 Airy Unit, Gain 791; Channel A488 Pinhole 1.6 Airy Unit, Gain 659; Channel A647 Pinhole 1.4 Airy Unit, Gain 719.</li>
	</ol>
	</li>
	<li>After adjusting the focus of the single channels, proceed to acquire the images at a 1,024 x 1,024 frame size and a 12.61 &#181;s pixel dwell.</li>
	<li>Count the percentage of MyoD-positive cells (control for the correct location under the lamina) that are LC3-negative and the percentage of MyoD-positive cells that display an LC3 signal. 
	NOTE: The percentage of MyoD-positive cells that exhibit LC3 staining is the readout of this protocol.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+autophagy+as+a+potential+mechanism+for+the+improved+physiological+function+by+simvastatin+in+muscular+dystrophy.">Enhanced autophagy as a potential mechanism for the improved physiological function by simvastatin in muscular dystrophy.</a> Autophagy. 12 (4), 705-706 (2016).</li><li>Whitehead, N. 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=A+new+therapeutic+effect+of+simvastatin+revealed+by+functional+improvement+in+muscular+dystrophy.">A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy.</a> Proc Natl Acad Sci U S A. 112 (41), 12864-12869 (2015).</li><li>Fiacco, E., 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=Autophagy+regulates+satellite+cell+ability+to+regenerate+normal+and+dystrophic+muscles.">Autophagy regulates satellite cell ability to regenerate normal and dystrophic muscles.</a> Cell Death Differ. 23 (11), 1839-1849 (2016).</li><li>Lee, I. H., 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=A+role+for+the+NAD-dependent+deacetylase+Sirt1+in+the+regulation+of+autophagy.">A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.</a> Proc Natl Acad Sci U S A. 105 (9), 3374-3379 (2008).</li><li>Rubinsztein, D. C., Mari&#241;o, G., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+aging.">Autophagy and aging.</a> Cell. 146 (5), 682-695 (2011).</li><li>Colman, R. J., 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=Caloric+restriction+delays+disease+onset+and+mortality+in+rhesus+monkeys.">Caloric restriction delays disease onset and mortality in rhesus monkeys.</a> Science. 325 (5937), 201-204 (2009).</li><li>Levine, B., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+pathogenesis+of+disease.">Autophagy in the pathogenesis of disease.</a> Cell. 132 (1), 27-42 (2008).</li><li>Yang, 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=Long-Term+Calorie+Restriction+Enhances+Cellular+Quality-Control+Processes+in+Human+Skeletal+Muscle.">Long-Term Calorie Restriction Enhances Cellular Quality-Control Processes in Human Skeletal Muscle.</a> Cell Rep. 14 (3), 422-428 (2016).</li><li>Wenz, T., 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=Increased+muscle+PGC-1alpha+expression+protects+from+sarcopenia+and+metabolic+disease+during+aging.">Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging.</a> Proc Natl Acad Sci USA. 106 (48), 20405-20410 (2009).</li><li>Carnio, S., 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=Autophagy+Impairment+in+Muscle+Induces+Neuromuscular+Junction+Degeneration+and+Precocious+Aging.">Autophagy Impairment in Muscle Induces Neuromuscular Junction Degeneration and Precocious Aging.</a> Cell Rep. , (2014).</li><li>Sandri, M., 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=Misregulation+of+autophagy+and+protein+degradation+systems+in+myopathies+and+muscular+dystrophies.">Misregulation of autophagy and protein degradation systems in myopathies and muscular dystrophies.</a> J Cell Sci. 126 (Pt 23), 5325-5333 (2013).</li><li>Cerletti, M., 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=Short-term+calorie+restriction+enhances+skeletal+muscle+stem+cell+function.">Short-term calorie restriction enhances skeletal muscle stem cell function.</a> Cell Stem Cell. 10 (5), 515-519 (2012).</li><li>Gopinath, S. 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=FOXO3+promotes+quiescence+in+adult+muscle+stem+cells+during+the+process+of+self-renewal.">FOXO3 promotes quiescence in adult muscle stem cells during the process of self-renewal.</a> Stem Cell Reports. 2 (4), 414-426 (2014).</li><li>Tang, A. 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=Induction+of+autophagy+supports+the+bioenergetic+demands+of+quiescent+muscle+stem+cell+activation.">Induction of autophagy supports the bioenergetic demands of quiescent muscle stem cell activation.</a> EMBO J. 33 (23), 2782-2797 (2014).</li><li>Garcia-Prat, 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=Autophagy+maintains+stemness+by+preventing+senescence.">Autophagy maintains stemness by preventing senescence.</a> Nature. 529 (7584), 37-42 (2016).</li><li>Klionsky, D. J., 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=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(3rd+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).</a> Autophagy. 12 (1), 1-222 (2016).</li></ol>

The authors have nothing to disclose

This protocol describes how to monitor autophagy in skeletal muscle stem cells during compensatory muscle regeneration. Several antibodies for the co-staining of LC3 and MyoD were tried, and the ones that work in mouse tissue sections and create successful results are listed here (see Materials Table). The permeabilization with methanol (see step 3.2.2) is highly recommended for successful staining.
The limitation of this protocol is linked to the intrinsic variability of the ...

Skeletal muscle regeneration is the result of the interaction between adult stem cells (Muscle Satellite Cells, MuSCs) and other cell types that are involved in the regenerative process. Muscle homeostasis and functionality are maintained by the combined signals arising from the muscle niche and systemic cues1,2. Throughout the lifetime, changes in the MuSC functionality, the muscle niche, and the systemic cues have been reported, leading to the decline of functional capacities in the elderly3. MuSCs are set in a niche beneath the basal lamina and, upon muscle injury, are activated to repair damaged muscles4,5. In order to ensure a productive regenerative response, it is crucial that MuSCs coordinate different processes necessary for the exit from quiescence, the self-renewal, and the proliferative expansion stage followed by the myogenic differentiation6. In the elderly and in muscular chronic diseases, all these functions are compromised, leading to altered muscle functionality2,3,6,7,8,9,10,11,12,13.
Macroautophagy (referred hereafter as autophagy) is emerging as a crucial biological process essential to preserve tissue homeostasis14. The autophagic process encloses trafficking mechanisms, where portions of cytoplasm, organelles, and proteins are engulfed into vesicles that eventually are degraded via the lysosome pathway, promoting the removal of toxic molecules and the recycling of macromolecules. This provides energy-rich compounds to support cell and tissue adaptation under stress or other adverse conditions15,16. Together with its cell-survival activity, autophagy can also work as a cell-death inducer, depending upon cell tissue context (e.g., normal versus cancer tissue) and the type of stress stimulus17,18.
Recent evidence indicates that autophagy is required to maintain muscle mass and myofiber integrity19,20 and has been reported to be impaired in different muscle dystrophies21,22,23, including Duchenne Muscular Dystrophy (DMD)24,25,26,27,28,29,30.Likewise, a progressive reduction of the autophagic process has been observed in the elderly31,32,33,34,35, after a loss of muscle mass (referred as sarcopenia)32,33,34,35,36,37, and in myofiber survival38.
A close relationship between autophagy and the regenerative potential of skeletal muscles was anticipated by a study from Wagers&#39;s laboratory, which showed that a calorie restriction enhances MuSC availability and activity39. This notion was further supported by the recent observation that the Foxo3-Notch axis activates the autophagic process during self-renewal40 and the MuSC transition from the quiescent to the proliferating state41. These data agree with the progressive reduction of basal autophagy from young to old and geriatric MuSCs, in association with the numerical and functional decline of MuSCs during ageing42.
In a recent paper, we demonstrated a close relationship between autophagy and the compensatory muscle regeneration that distinguishes the early stages of DMD progression. Accordingly, we observed a reduced autophagic flux at later stages of disease progression, when muscle regeneration is compromised and fibrotic tissue deposition occurs. Intriguingly, we showed that, in regenerating conditions, autophagy is activated in MuSCs and that modulating the autophagic process impacts MuSC activation and functionality30.
Altogether, these data highlight the urgency to explore the autophagic process in MuSCs during muscle regeneration in normal and pathological conditions and throughout the life span. Here, we provide a protocol to monitor the autophagic process in MuSCs in muscle regenerative conditions by performing in situ immunostaining for microtubule-associated protein 1A/1B-light chain 3 (LC3), a marker of autophagy43, and MyoD, a marker of myogenic lineage, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC, which plays a key role in orchestrating muscle regeneration.

<table><tbody><tr><td>C57BL/6J</td><td>The Jackson Laboratory</td><td>000664</td><td>WT mice</td></tr><tr><td>Cardiotoxin 1</td><td>Latoxan</td><td>L8102</td><td></td></tr><tr><td>Millex-VV</td><td>Merck Millipore</td><td>SLVV033RS</td><td>Syringe Filter Unit, 0.1 &amp;micro;m, PVDF, 33 mm, gamma sterilized</td></tr><tr><td>Chloroquine diphosphate salt</td><td>Sigma-Aldrich</td><td>C6628</td><td>Caution:&lt;br/&gt; Harmful if swallowed</td></tr><tr><td>BD Micro-Fine + 0.5 mL</td><td>BD</td><td>324825</td><td></td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura Finetek</td><td>25608-930</td><td></td></tr><tr><td>Tissue-Tek Cryomold Intermediate</td><td>Sakura Finetek</td><td>4566</td><td></td></tr><tr><td>2-Methylbutane</td><td>Sigma-Aldrich</td><td>277258</td><td></td></tr><tr><td>Hematoxylin Solution, Harris Modified</td><td>Sigma-Aldrich</td><td>HHS32</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma-Aldrich</td><td>HT110132</td><td></td></tr><tr><td>o-Xylene</td><td>Sigma-Aldrich</td><td>X1040</td><td>Caution:&lt;br/&gt; Flammable liquid and vapour; May be fatal if swallowed and enters airways; Harmful in contact with skin; May cause respiratory irritation; Causes serious eye irritation</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148</td><td>Caution:&lt;br/&gt; Flammable solid; Harmful if swallowed; Causes skin irritation; May cause an allergic skin reaction; Causes serious eye damage; May cause respiratory irritation; Suspected of causing cancer</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Eukitt - Quick-hardening mounting medium</td><td>Sigma-Aldrich</td><td>3989</td><td></td></tr><tr><td>AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>115-007-003</td><td></td></tr><tr><td>LC3B Antibody</td><td>Cell signaling Technology</td><td>2775</td><td></td></tr><tr><td>Monoclonal mouse anti-MyoD&lt;br/&gt; (concentrated) clone 5.8A</td><td>DAKO - Agilent Pathology Solutions</td><td>M3512</td><td></td></tr><tr><td>Laminin-2 (&amp;alpha;-2-chain) monoclonal antibody</td><td>Enzo Life Sciences</td><td>4H8-2</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)</td><td>Life technologies</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 594 Goat Anti-Mouse IgG (H+L)</td><td>Life technologies</td><td>A11005</td><td></td></tr><tr><td>Alexa Fluor Goat Anti-Rat IgM Antibody</td><td>Life technologies</td><td>A21248</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr></tbody></table>

in situ immunofluorescent staining of autophagy in muscle stem cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

This protocol describes an efficient in situ method to detect autophagy in MuSCs during muscle regeneration.
CTX In Vivo Treatments:
Use CTX to induce muscle damage in TA muscles and use unperturbed muscles as controls. Since autophagy is highly dynamic, block the autophagic flux by performing IP injections of CLQ (Figure 1)...

Watch this Scientific Journal Video about In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells at JoVE.com

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

in-situ-immunofluorescent-staining-of-autophagy-in-muscle-stem-cells

Research

JoVE Journal

Biology

10.1K Views.  Italian National Research Council.  Actieve autofagie is geassocieerd met productieve spierregeneratie, die essentieel is voor het activeren van spierstamcellen (MuSC). Hier bieden we een protocol voor de in situ detectie van LC3, een autofagie marker in MyoD-positieve MuSCs van spierweefsel secties van controle en gewonde muizen. 

Video: In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

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 ...

Developmental Biology

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, ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

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

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

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

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

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

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

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

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

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

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

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

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

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.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Harvesting Mouse Skeletal Muscle to Culture Stem Cells and Their Niche

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Author Spotlight: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components 

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

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