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.
