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 border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:332px;">C57BL/6J</td>
			<td style="width:251px;">The Jackson Laboratory</td>
			<td style="width:128px;">000664</td>
			<td style="width:196px;">WT mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cardiotoxin 1</td>
			<td>Latoxan</td>
			<td>L8102</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Millex-VV</td>
			<td>Merck Millipore</td>
			<td>SLVV033RS</td>
			<td style="width:196px;">Syringe Filter Unit, 0.1 &#181;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Chloroquine diphosphate salt</td>
			<td>Sigma-Aldrich</td>
			<td>C6628</td>
			<td style="width:196px;">Caution: 
			Harmful if swallowed</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">BD Micro-Fine + 0,5 mL</td>
			<td>BD</td>
			<td>324825</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue-Tek O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue-Tek Cryomold Intermediate</td>
			<td>Sakura Finetek</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2-Methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>277258</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hematoxylin Solution, Harris Modified</td>
			<td>Sigma-Aldrich</td>
			<td>HHS32</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Eosin Y solution, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr height="122">
			<td height="122" style="height:122px;">o-Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>X1040</td>
			<td style="width:196px;">Caution: 
			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 height="136">
			<td height="136" style="height:136px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td style="width:196px;">Caution: 
			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 height="17">
			<td height="17" style="height:17px;">DPBS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Eukitt - Quick-hardening mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-007-003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LC3B Antibody</td>
			<td>Cell signaling Technology</td>
			<td>2775</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:332px;">Monoclonal mouse anti-MyoD 
			(concentrated) clone 5.8A</td>
			<td>DAKO - Agilent Pathology Solutions</td>
			<td>M3512</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Laminin-2 (&#945;-2-chain) monoclonal antibody</td>
			<td>Enzo Life Sciences</td>
			<td>4H8-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A11008</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor 594 Goat Anti-Mouse IgG (H+L)</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A11005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor Goat Anti-Rat IgM Antibody</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A21248</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">DAPI (4&#39;,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. 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.

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

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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 lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

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

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...