Medicine
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Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development
Chapters
Summary July 1st, 2020
Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin Δ8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).
Transcript
Many protocols exist for isolating single myofibers from the skeletal muscles of adult mice. Our protocol is devised for very young mice and for small mice with a very fragile myofibers. This standardized procedure can be used for studying myofiber-assisted muscle stem cells during postnatal development or in mouse models of diseases that make myofibers particularly susceptible to mechanical stress.
The procedure will be demonstrated by Gloria Pegoli, a PhD student, and Federica Lucini, a post-doctorate, both from a laboratory. For muscle dissection and harvest, use a pin to fix one hind limb from a euthanized, very young or very small mouse onto a dissecting board, and use sharp tweezers to lift the lower tendon of the tibialis anterior to ankle height. Cut the tendon and use fine scissors to cut all the way around the muscle to the tendon at the level of the patella.
Place the muscle into a tube of digestion solution and lift the lower tendon of the extensor digitorum longus, pulling gently upward on the tendon to separate the extensor digitorum longus from other muscles. Cut and harvest the muscle. Rotate the leg to view the back muscles and lift the achilles tendon.
A gastrocnemius muscle will automatically separate from the other muscles. Cut the upper tendon at the back of the patella and place the muscle in the digestion solution. Lift the external tendon of the leg and roll the tweezers under the tendon to gently separate the tendon from the muscle.
Then, cut to harvest the muscle and collect the same muscles from the other leg in the same manner. When all of the muscles have been collected, place the collection tube into a 37 degree celsius water bath for 45 to 50 minutes and vigorously invert the tube 10 times every 10 minutes. When the muscles begin to loosen up and the myofibers become visible, shake the samples one last time before carefully transferring the digestion suspension into a pre-warmed or serum coated 100-millimeter Petri dish containing 10 milliliters of washing solution.
To isolate single myofibers, place the dish under a dissecting microscope and use a horse serum coated 200-microliter pipette tip to pick apart the individual muscle fibers. Transfer the viable myofibers into a horse serum coated 35-millimeter Petri dish containing five milliliters of pre-warmed washing solution. When all of the viable myofibers have been collected, equilibrate the myofiber samples in the cell culture incubator for five minutes.
If more myofibers are needed, use a large-bore glass pipette to triturate the remaining muscle tissue sample until additional fibers are mechanically released. When enough myofibers have been collected, place the dish into the cell culture incubator for one hour for an additional equilibration period. At the end of the incubation, transfer individual myofibers to a new, pre-warmed horse serum coated dish containing the appropriate culture medium and return the plate to the cell culture incubator.
For an immunofluorescent analysis of the myofibers, after cross linking, remove all but enough supernatant, not to remove any fibers and add to the same tube one milliliter of 0.5%Triton X-100 in PBS for a five-minute incubation with gentle agitation. At the end of the incubation, allow the fibers to settle for five minutes before replacing the supernatant with 1.5 milliliters of PBS. After five minutes of gentle agitation, allow the fibers to settle for another five minutes before replacing the supernatant with one milliliter of blocking solution.
After one hour of gentle agitation, replace the blocking solution with fresh blocking solution containing primary antibodies of interest for an overnight incubation at four degrees celsius with gentle agitation. The next morning, wash the fibers with three five-minute washes in one milliliter of fresh 0.25%Tween 20 in PBS using gentle agitation and five minutes of sedimentation at room temperature for wash. After the last wash, label the fibers with the appropriate fluorochrome conjugated secondary antibodies diluted in blocking solution for one hour at room temperature with gentle agitation, protected from light, followed by three washes in PBS plus 0.1%Tween, protected from light, as demonstrated.
After the last wash, replace the supernatant with one milliliter of DAPI solution for a five-minute incubation at room temperature with gentle agitation, protected from light. At the end of the incubation, wash the fibers and use a blocking solution coated pipette with a cut tip to collect the fibers and carefully spread the fibers on a glass slide. Place the slide under a dissecting microscope, and using only the natural light reflected by the mirror, use a new 200 microliter pipette tip to gently spread the fibers and to remove the excess solution.
After removing the excess solution, allow the slide to dry in the dark for 10 to 15 minutes until only a very small volume of solution remains. Then, add an appropriate volume of mounting medium on the fibers before carefully placing a cover slip over the tissues. To retrieve a sufficient number of long, viable fibers that can survive 96 hours in growth factor rich medium, four different muscles are typically harvested and digested.
Only the most intact fibers should be transferred to the culture medium. Broken fibers and other debris should be discarded. After 72 hours of culture, activated satellite cells generate cell aggregates bound to the myofiber.
We observe that the cell culture's derived from Lamin delta eight 11 double negative mice are formed by a smaller number of cells. After 96 hours, satellite cells are expressing MyoG, become visible within the cell cluster, initiating the differentiation toward new myofibers. Notably, a delayed dynamics of satellite cell differentiation is observed in homozygous mutant Lamin knockout mice compared to their wild type counterparts.
Be sure to practice the muscle dissection steps as a quick and precise muscle harvest is essential to the success of the experiment. This technique allow the tracking of single satellite cells during activation and differentiation, facilitating the study of key processes underlying muscle growth and regeneration under different physiological and pathological conditions.
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