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-buffered saline (PBS) in a 35 mm Petri dish for muscle collection.</li>
	<li>Sacrifice the mouse by cervical dislocation or by using CO2 and sterilize the skin with ethanol. Remove the skin from the hindlimbs and upper limbs by using scissors and tweezers, to facilitate the isolation of muscles. 
	NOTE: Muscles can be isolated from neonatal or adult mice. Neonatal mice have an increased number of satellite cells compared to adult mice. However, the total number of satellite cells that can be isolated from a single adult mouse is sufficient to perform the experiment described below.</li>
	<li>Isolate tibialis, soleus, extensor digitorum longus (EDL), gastrocnemius, quadriceps, and triceps, and put them in the Petri dish containing PBS. Leave the dish on ice. Carefully remove tendons and fat with sterilized scissors and tweezers.</li>
</ol>
2. Isolation of primary myoblasts
NOTE: All the procedures for cell culture are done in sterile conditions.
<ol>
	<li>Remove the muscles from the PBS. Cut and mince the muscles, by using sterile curved scissors, until a uniform mass is obtained. Put the muscle pieces into a 50 mL tube.</li>
	<li>Add 10 mL of the digestion medium to the muscle pieces and incubate at 37 &#176;C for 30 min under strong agitation (250 min-1) in a water bath, to enzymatically digest the muscles.</li>
	<li>Add 10 mL of Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) containing 10% fetal bovine serum (FBS), 1% glutamine, 1% penicillin/streptomycin, and 1% gentamicin (blocking medium) to stop the digestion.</li>
	<li>Centrifuge at 40 x g for 5 min and collect the supernatant in a 50 mL tube. Save the pellet on ice. 
	NOTE: After the centrifugation, the supernatant contains some satellite cells, blood cells, endothelial cells, and fibroblasts, while the pellet contains pieces of undigested muscle and connective tissue. To increase the number of isolated cells, the pellet must be subjected to additional steps of digestion as described below.</li>
	<li>Centrifuge the supernatant at 650 x g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of DMEM containing 10% FBS. Keep the resuspended pellet on ice.</li>
	<li>Repeat steps 2.2&#8211;2.5 2x for the rest of the pellet obtained in step 2.4 and add the subsequently resuspended pellets to the first resuspended pellet conserved on ice.</li>
	<li>Pass the resuspended pellets through a 70 &#181;m filter and then through a 40 &#181;m filter. Add 15 mL of DMEM with 10% FBS, and centrifuge at 650 x g for 5 min.</li>
	<li>Resuspend the cell pellet in 3 mL of red blood cell lysis buffer to deplete the red blood cells. Incubate it for 5 min at room temperature. Add 40 mL of PBS to stop the lysis of the red blood cells, and centrifuge at 650 x g for 5 min. Remove the supernatant and resuspend the pellet in 5 mL of DMEM with 10% FBS.</li>
	<li>As preplating step, plate the cells in 20 mL of proliferation medium in a 150 mm uncoated Petri dish. After 1 h of incubation in a cell culture incubator (37 &#176;C, 5% CO2), collect the medium. 
	NOTE: Fibroblasts, attaching to the plate, are then discarded, while satellite cells remain in suspension.</li>
	<li>Repeat step 2.9 3x and, at the last preplating step, collect the medium which contains the satellite cells in suspension.</li>
	<li>Centrifuge at 650 x g for 5 min.</li>
	<li>While the cells are centrifuging, coat two 150 mm Petri dishes with collagen (10 mL of a 0.1% solution in 0.1 M acetic acid). To do so, put the collagen on the plate, incubate for 5 min, and remove it. Let the plate dry for 30 min.</li>
	<li>Discard the supernatant obtained in step 2.11 and resuspend the cell pellet in 40 mL of proliferation medium (Table of Materials). Plate the cells on two collagen-coated 150 mm Petri dishes (20 mL per dish) and keep the cells in a cell culture incubator (37 &#176;C, 5% CO2, 5% O2) in proliferation medium for 2&#8211;3 days with 1 &#181;g/mL of doxycycline to induce H2B-GFP expression. 
	NOTE: This step allows the proliferation of myoblasts to obtain a higher number of cells for further assays. Cell density is maintained very low to avoid a spontaneous differentiation of myoblasts into myotubes.</li>
</ol>
3. Myoblast proliferation and differentiation assays
NOTE: When myoblast density is high, some cells initiate elongation, and then, it is necessary to split the cells. Generally, it is possible to split cells 2x&#8211;3x without affecting their phenotype.
<ol>
	<li>Discard the medium, wash the cells with 5 mL of PBS, add 2 mL of trypsin (1x), and incubate the plate at 37 &#176;C in a cell incubator for 5 min. Check under the microscope and, when round cells detach, add 5 mL of DMEM with 10% FBS to inactivate the trypsin. Count the number of cells (usually it is possible to obtain about 1 x 106 cells/mouse). 
	NOTE: Incubation with trypsin for 5 min is usually enough to detach the proliferating myoblasts.</li>
	<li>Subject the cells to one of the assays described below.
	<ol>
		<li>Proliferation assay 
		<ol>
			<li>Plate 50,000 cells/well in 1 mL/well of proliferation medium in a collagen-coated 12-well plate and incubate the plated cells in a cell culture incubator (37 &#176;C, 5% CO2). Fix and count the cells at 24, 48, or 72 h. 
			NOTE: The medium is replaced every 48 h to avoid nutrient exhaustion.</li>
		</ol>
		</li>
		<li>Differentiation assay 
		<ol>
			<li>Coat a 12-well plate with 350 &#181;L of differentiation medium containing basement membrane matrix (1:100). Incubate the plate at 37 &#176;C for 30 min and remove the medium.</li>
			<li>Plate 200,000 cells/well in the differentiation medium in the matrix-coated plate. Incubate the cells in a cell culture incubator (37 &#176;C, 5% CO2, 5% O2) until they adhere to the plate (2 h are generally enough for the cells to adhere and to start differentiation).</li>
			<li>To assess differentiation, perform staining for myosin heavy chain and nuclei as previously described11. Alternatively, perform live-imaging analyses as described below.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Live-imaging of myoblast differentiation and nuclear tracking
<ol>
	<li>For live cell imaging, put the cells, obtained in section 3.2.2 and plated in a 12-well plate, under a confocal microscope, equipped with an incubation system to maintain the cells at 37 &#176;C in 5% CO2.</li>
	<li>Use a 20x dry objective (0.7 NA) to get fields of view with hundreds of cells, enough to monitor myofiber formation. H2B-GFP cells can be imaged with a low-intensity 488 nm Argon laser. 
	NOTE: An open pinhole ensures that the image depth (~10 &#181;m) contains the cell thickness so that cells are kept in focus throughout the experiment. Using a confocal microscope is advantageous since it improves the signal-to-noise ratio of the images, although wide-field microscopy could also be used. Myofiber formation can be monitored through the transmission channel.</li>
	<li>Acquire images of 16-bit and 1,024 x 1,024 pixels per frame every 6 min for 16 h. For each acquired position, generate a multiframe.tif file (see example in the Supplementary Files). 
	NOTE: In the present protocol, commercial software associated with the microscope (Table of Materials) has been used; use the appropriate software to achieve the same goal when using other microscopes.</li>
	<li>Download the software provided as a .zip supplementary file. 
	NOTE: The routines are scripts that must be run in MATLAB. The software is an adaptation of a published software12 optimized for the tracking of the nuclei during myotube formation. This software is divided into two parts: the first one identifies the nuclei in each frame by segmenting them, while the second one generates &#8220;tracks&#8221; (trajectories) of nuclei movement. The complete code for nuclear segmentation and tracking and a step-by-step guide to getting started are provided in the Supplementary Files.</li>
	<li>Extract the zip file and save it in a desired path on the personal computer (PC) in use. Perform all the below passages on a PC with the necessary software already installed. Note that the .zip file is composed of three folders as mentioned below. 
	NOTE: Segmentation Routines contains the functions to be run for the segmentation. Tracking Routines, instead, contains the functions required for the tracking. Example Segmentation Tracking provides the basic scripts and files to get acquainted with the system. The software used for segmentation and tracking of the nuclei runs properly in MATLAB, R2015a or later versions.</li>
	<li>To segment the nuclei from the .tif file, name the file, for example, NameFile.tif.
	<ol>
		<li>Open the Example Segmentation Tracking folder and click on DoSegmentation.m. This will open the command window in MATLAB.</li>
		<li>Check the Current Folder window on the left to see all the elements in the Example of Segmentation Tracking folder. Double-click on DoSegmentation.m. 
		NOTE: Detailed information on the modifications that have to be done in the script can be found in the StepbyStep.txt file. It is mandatory to modify the script so that the variable filenameinput is NameFile.tif and folderinput is the folder where the .tif file is contained.</li>
		<li>Run the script (by clicking on the green Run button). The result of the segmentation will be saved as a file.mat (SegmentedNameFile.mat), while the segmentation of the nuclei can be visualized in the output figure. 
		NOTE: Parameters for the segmentation, described in the script, can be modified if needed. The quality of the segmentation can be checked using the script CheckSegmentation.m (see the StepbyStep.txt file). Based on this, if the quality of the segmentation is poor (only a few nuclei detected), adjust the parameters of the segmentation, clearly indicated in the DoSegmentation.m script, and repeat the procedure.</li>
	</ol>
	</li>
	<li>To generate the tracks (i.e., the trajectories of each of the nuclei in time), using the nuclear positions obtained in the segmentation, run the script GenerateTracks.m in the same working folder. Be sure to add the proper segmentation file as an input, obtained before (see the StepbyStep.txt file for further information). 
	NOTE: As an output, the user will obtain a file TrackedNameFile.mat, with a matrix for the x-coordinates and another matrix for the y-coordinates of the generated tracks.</li>
	<li>Evaluate the quality of the tracks using CheckTracking.m (see the StepbyStep.txt file for further information). Use the output figure of this last passage to help visualize each nuclei position in time. Check on the left for green crosses that indicate each segmented nucleus. To select one specific nucleus, use the lower scrollbar. Note that the selected nucleus will be circled in red, while on the right, the trajectory of the selected cell can be followed in time (by using the upper scrollbar).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dynamics+in+the+muscle+satellite+cell+niche.">Cellular dynamics in the muscle satellite cell niche.</a> EMBO reports. 14 (12), 1062-1072 (2013).</li><li>Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cell+of+skeletal+muscle+fibers.">Satellite cell of skeletal muscle fibers.</a> The Journal of Biophysical and Biochemical Cytology. 9, 493-495 (1961).</li><li>Motohashi, N., Asakura, Y., Asakura, 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,+culture,+and+transplantation+of+muscle+satellite+cells.">Isolation, culture, and transplantation of muscle satellite cells.</a> Journal of Visualized Experiments. 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(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. 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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>

The authors have nothing to disclose.

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...

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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 ...

exploiting-live-imaging-to-track-nuclei-during-myoblast

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Fibroblast Transformation and Muscle Stem Cells

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8.0K Views.  Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute. 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.

Video: Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

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Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

Determining the Contribution of the Energy Systems During Exercise

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

4D Imaging of Protein Aggregation in Live Cells

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression