This work was supported by the AFM-Telethon to E.V. (#21545) and by the Ospedale San Raffaele (OSR) Seed Grant to S.Z. (ZAMBRA5X1000). Dr. Jean-Yves Tinevez from the Image Analysis Hub of the Institut Pasteur is acknowledged for publicly sharing his &#34;Simple Tracker&#34; MATLAB routines.

All procedures involving animal subjects were approved by the San Raffaele Institutional Animal Care and Use Committee.
1. Dissection of mouse hindlimb muscles
<ol>
	<li>Sterilize tweezers and scissors (both straight and curved) by autoclaving.</li>
	<li>Prepare and filter (0.22 &#181;m) all the media (blocking medium, digestion medium, proliferating medium, and differentiating medium) before starting the experiment (see Table of Materials).</li>
	<li>Put 5 mL of phosphate-b...

<ol>
	<li>Janssen, I., Heymsfield, S. B., Wang, Z. M., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+mass+and+distribution+in+468+men+and+women+aged+18-88+yr.">Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr.</a> Journal of Applied physiology. 89 (1), 81-88 (2000).</li><li>Ten Broek, R. W., Grefte, S., Von den Hoff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+factors+and+cell+populations+involved+in+skeletal+muscle+regeneration.">Regulatory factors and cell populations involved in skeletal muscle regeneration.</a> Journal of Cellular Physiology. 224 (1), 7-16 (2010).</li><li>Bentzinger, C. F., Wang, Y. X., Dumont, N. A., Rudnicki, M. 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(86), e50846 (2014).</li><li>Pasut, A., Jones, A. E., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+individual+myofibers+and+their+satellite+cells+from+adult+skeletal+muscle.">Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle.</a> Journal of Visualized Experiments. (73), e50074 (2013).</li><li>Soriano-Arroquia, A., Clegg, P. D., Molloy, A. P., Goljanek-Whysall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Culture+of+Myogenic+Precursor+Cells/Primary+Myoblasts+from+Skeletal+Muscle+of+Adult+and+Aged+Humans.">Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans.</a> Journal of Visualized Experiments. (120), e55047 (2017).</li><li>Roman, W., Gomes, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+positioning+in+skeletal+muscle.">Nuclear positioning in skeletal muscle.</a> Seminars in Cell &amp; Developmental Biology. , (2017).</li><li>Pimentel, M. R., Falcone, S., Cadot, B., Gomes, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Differentiation+of+Mature+Myofibers+for+Live+Imaging.">In Vitro Differentiation of Mature Myofibers for Live Imaging.</a> Journal of Visualized Experiments. (119), e55141 (2017).</li><li>Foudi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+histone+2B-GFP+retention+reveals+slowly+cycling+hematopoietic+stem+cells.">Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells.</a> Nature Biotechnology. 27 (1), 84-90 (2009).</li><li>Tirone, 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=High+mobility+group+box+1+orchestrates+tissue+regeneration+via+CXCR4.">High mobility group box 1 orchestrates tissue regeneration via CXCR4.</a> The Journal of Experimental Medicine. 215 (1), 303-318 (2018).</li><li>Zambrano, S., De Toma, I., Piffer, A., Bianchi, M. E., Agresti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+oscillations+translate+into+functionally+related+patterns+of+gene+expression.">NF-kappaB oscillations translate into functionally related patterns of gene expression.</a> eLife. 5, e09100 (2016).</li><li>Adamski, 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=Effects+of+Hoechst+33342+on+C2C12+and+PC12+cell+differentiation.">Effects of Hoechst 33342 on C2C12 and PC12 cell differentiation.</a> FEBS Letters. 581 (16), 3076-3080 (2007).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Transactions on Systems, Man, and Cybernetics. 9 (1), 62-66 (1979).</li><li>. Simpel Tracker - File Exchange - MATLAB Central Available from: <a target="_blank" href="https://it.mathworks.com/matlabcentral/fileexchange/34040-simple-tracker">https://it.mathworks.com/matlabcentral/fileexchange/34040-simple-tracker</a> (2016)</li><li>Burkard, R., Dell'Amico, M., Martello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assignment Problems, Revised Reprint. , (2009).</li><li>Falcone, 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=N-WASP+is+required+for+Amphiphysin-2/BIN1-dependent+nuclear+positioning+and+triad+organization+in+skeletal+muscle+and+is+involved+in+the+pathophysiology+of+centronuclear+myopathy.">N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy.</a> EMBO Molecular Medicine. 6 (11), 1455-1475 (2014).</li><li>Syverud, B. C., Lee, J. D., VanDusen, K. W., Larkin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Purification+of+Satellite+Cells+for+Skeletal+Muscle+Tissue+Engineering.">Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering.</a> Journal of Regenerative Medicine. 3 (2), (2014).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nature Protocols. 10 (10), 1612-1624 (2015).</li></ol>

Muscle fibers (myofibers) are multinucleated cells that are formed by the fusion of muscle precursors cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation2,3,4. To assess myoblast differentiation, the common procedure consists of culturing myoblasts in differentiating medium and fixing the cells at different time points to perform immunofluorescence staining for MyHC, ...

Skeletal muscle is the largest tissue in the human body, totaling 35%-40% of body mass1. Satellite cells are muscle stem cells, anatomically characterized by their position (juxtaposed to the plasma membrane, underneath the basal lamina of muscle fibers), that give rise to proliferating myoblasts (myogenic progenitor cells), which eventually differentiate and integrate into existing myofibers and/or fuse to form new myofibers2,3,4. Their discovery and the progress in the study of their biology has led to significant insights into muscle development and regeneration.
Protocols to isolate and differentiate myoblasts into myotubes have been developed many years ago and are still widely used to study skeletal muscle differentiation5,6,7. However, most of these methods represent static procedures that rely on the analysis of fixed cells and, consequently, do not allow scientists to fully explore highly dynamic processes, such as myoblast fusion and myofiber maturation. The most striking example is nuclear positioning, which is tightly regulated, with nuclei initially in the center of the myofiber and, then, located at the periphery after myofiber maturation8,9. Live imaging is the most appropriate technique to obtain further insights into such a peculiar phenomenon.
Here, we describe a method that enables scientists to record myoblast differentiation and myotube formation by time-lapse microscopy and to perform quantitative analyses from the automatic tracking of myoblast nuclei. This method provides a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior during differentiation and fusion. The protocol is divided into four different parts, namely (1) the collection of muscles from the hindlimbs of mice, (2) the isolation of primary myoblasts that consists in mechanical and enzymatic digestion, (3) myoblast proliferation and differentiation, and (4) live imaging to track nuclei within the first 16 h of myoblast differentiation.
In the following procedure, myoblasts are isolated from H2B-GFP mice and treated with 1 &#181;g/mL of doxycycline to induce H2B-GFP expression, as previously described10. Alternatively, it is possible to isolate the myoblasts from other transgenic mice that express a fluorescent protein in the nucleus or to transfect the cells isolated from wild-type mice to express a fluorescent protein in the nucleus, as described by Pimentel et al.9.

<table>
	<tbody>
		<tr>
			<td>chicken embryos extract</td>
			<td>Seralab</td>
			<td>CE-650-J</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Sigma</td>
			<td>C9263-1G</td>
			<td>125units/mg</td>
		</tr>
		<tr>
			<td>collagen from calf skin</td>
			<td>Sigma</td>
			<td>C8919</td>
			<td></td>
		</tr>
		<tr>
			<td>dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.78 units/mg</td>
		</tr>
		<tr>
			<td>doxyciclin</td>
			<td>Sigma</td>
			<td>D1515</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Life technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>Sigma</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Invitrogen</td>
			<td>16050-098</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>life technologies</td>
			<td>33342</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Sigma</td>
			<td>I3390</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>red blood cells lysis medium</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>40 mg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dispase</td>
			<td>70 mg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>20 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>filtered 0.22um</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>10%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutammine</td>
			<td>1%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>1%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>1 &#8240;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>filtered 0.22um</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>proliferation medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>20%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutammine</td>
			<td>1%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>1%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>1 &#8240;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>chichen embryo extract</td>
			<td>3%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>filtered 0.22um</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>differentiation medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>2%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutammine</td>
			<td>1%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>1%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>1 &#8240;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>chichen embryo extract</td>
			<td>1%</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>filtered 0.22um</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

exploiting live imaging to track nuclei during myoblast differentiation and fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

To automatically follow nuclear movement during myoblast differentiation in live imaging, the nuclei should preferentially be fluorescently labeled. It is important to note that using DNA-intercalating molecules is not feasible because these molecules interfere with the proliferation and differentiation of primary myoblasts13. As an example, proliferation and differentiation have been analyzed in primary myoblasts cultured with or without Hoechst (<strong class="xf...

Watch this Scientific Journal Video about Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion at JoVE.com

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Without protocol, it is possible to investigate the dynamic process of myoblast differentiation and myofiber formation, in particular nuclear positioning by exploiting live cell imaging. Live cell imaging of myoblast with foreign cell nuclei allow the study of myoblast differentiation in living cells and the automatic tracking of the nuclei. Visual demonstration helps with understanding how to combine primary cell isolation with live imaging and imaging analysis.Demonstrating the procedure with Frederica Colombo, will be Giorga Careccia, a PhD student in our laboratory. Begin by harvesting the tibialis soleus, extensor digitorum longus, gastrocnemius, quadriceps, and triceps muscles from both hind limbs of an adult mouse into a Petri dish of PBS on ice. Use sterile scissors and tweezers to carefully remove the tendons and fat from each muscle and transfer the muscles into an empty Petri dish.Using sterile curved scissors cut and mince the muscles until a uniform mass is obtained and transfer the muscle pieces to a 50 milliliter tube. Then add 10 milliliters of digestion medium to the muscle pieces for a 30 minute incubation in a 37 degree celsius water bath at 250 rotations per minute. At the end of the enzymatic digestion, stop the reaction with 10 milliliters of blocking medium.And spin down the sample by centrifugation. Place the tube on ice and transfer the supernatant into a new 50 milliliter tube. Centrifuge the supernatant again.And re-suspend the pellet in one millimeter of DMEM. Supplement it with ten percent fetal bovine serum, or FBS, on ice. Digest the reserved pellet in 10 milliliters of fresh digestion medium as just demonstrated and combine the digested pellet with the reserve cell suspension, on ice.Strain the pulled cells through a 70 micrometer filter, followed by a 40 micrometer filter. And wash the cells in 15 millimeters of fresh DMEM, supplemented with 10 percent FBS. At the end of the centrifugation, re-suspend the pellet in three milliliters of red blood cell lysis buffer at room temperature.Stopping the lysis after five minutes with 40 milliliters of PBS and an additional centrifugation. Re-suspend the pellet in 20 milliliters of DMEM supplemented with 10 percent FBS and pre-plate the cells in uncoated Petri dish. After one hour at 37 degree celsius and five percent carbon dioxide collect the satellite cell containing supernatant.And pre-plate the cell suspension two more times as demonstrated. After the last plating, collect the satellite cells by centrifugation and re-suspend the pellet in 40 milliliters of fresh proliferation medium. The split the cells between two collagen coated 150 milliliters Petri dishes with one microgram per-milliliter of doxycycline per dish to induce H2BGFP expression.And return the cells to the cell culture incubator for two or three days. When the myoblasts have reached the appropriate experimental density, wash the cells in each dish with five milliliters of PBS and detach the cells from the plate bottoms with two milliliters of Trypsin per dish at 37 degree celsius for five minutes. Confirm detachment by light microscopy and stop the reaction with five milliliters of DMEM plus ten percent FBS per plate.For live cell imaging, plate two times ten to the fifth cells into each well of a 12 well plate coated with 350 microliters of differentiation medium, supplemented with basement membrane matrix per well for two hours. When the cells have adhered to the bottom of the plate, place the plate in a 37 degree celsius and five percent carbon dioxide incubator on a confocal microscope stage and use a 20 times dry objective to obtain fields of view with hundreds of cells to acquire 16 bit images with 1024 by 1024 pixels per frame every six minutes for 16 hours. For each acquired position, generate a multi frame dot tif file and download and extract the dot zip software provided in a selected folder.Open the example of segmentation tracking folder and click do segmentation dot M to open the command window in Matlab. Change the due segmentation dot M scripts so that the variable file name track is the name of the tif file and path input track is the folder where the dot tif is contained. Click run to run the script.The result of the segmentation will be saved as a file dot mat, while the segmentation of the nuclei can be visualized in the output figure. To generate the tracks, first double click generate tracks dot M in the same working folder. Next, change the file name to file name point the appropriate dot Mat file for the segmented nuclei.Click Run to run the script. The result of the tracking will be saved as another dot mat file and the generated tracks will be plotted. To evaluate the quality of the tracks.First double click the check tracking dot M and modify the file name to the appropriate dot tif name. Then click file name track to use the dot mat file of the tracks and click run. In the figure window use the lower scroll bar to select the nucleus.The selected nucleus will be circled in red, and the trajectory of the selected cell can be followed in time using the upper scroll bar. Green crosses indicate the detected nuclei. Proliferation and differentiation are strongly impaired in myoblasts cultured with Hoechst.Compared to myoblast's isolated from H2BGFP mice and cultured with doxycycline or wild type, myosin heavy chain labeled myoblasts. Live cell imaging with primary myoblasts expressing the H2BGFP protein allows tracking of the nuclei during differentiation. Merged images of transmission in GFP channels at the initial and final time points of the differentiation period, allow the identification of myotubes and consequently nuclei that end up integrating into myotubes or that do not fuse into myotubes.After imaging by confocal fluorescence microscopy, the nuclei can be segmented as demonstrated and watershed transform can be used to separate nearby objects. With this approach, most of the nuclei are identified in the image, allowing the nuclei motion to be tracked as demonstrated. For example, it appears that the total length of the trajectories is slightly higher for cells stained with Hoechst.Although the difference is not significant. However, computation of the total displacement during a time lapse reveals that the total displacement of the cells stained with Hoechst is significantly smaller than that of unstained cells. As predicted the total annual variation for the unstained cells is significantly smaller compared to cells stained with Hoechst.It is important to follow the protocol exactly as demonstrated in particular when combining the technical skills from the biological steps with the confocal microscope and software used steps. To study myoblast differentiation and nuclear positioning in another muscle model of interest it is possible to transfer isolated myoblast with fluorescent nuclear protein. This technique paved the way to explore cellular and nuclear dynamics during muscle differentiation and to further investigate defects in this processes.Under various pathological conditions such as Muscular dystrophies.

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8.0K visualizações.  Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute. Sem protocolo, é possível investigar o processo dinâmico de diferenciação myoblast e formação de miofibras, em particular o posicionamento nuclear explorando imagens de células vivas. Imagens de células vivas de míobios com núcleos celulares estranhos permitem o estudo da diferenciação myoblast em células vivas e o rastreamento automático dos núcleos. A demonstração visual ajuda a entender como combinar o isolamento celular primário com a análise de imagens e imagens ao vi...