I need to quantify the uptake of our therapeutic oligo drugs in the nuclei of muscle fibres isolated from adult mice. However these fibres have several satellite cells attached to them (see photo of an FDB fibre isolated from a 15wk old BL10 mouse) and therefore I need to remove them first in order to be certain that the oligo uptake measured in the nuclear fraction is from the muscle fibre nuclei only. Is there a way to remove or encourage migration of satellite cells quickly from the muscle fibres i.e. agitation, centrifugation etc?
The problem is that in order to get rid of satellite cells you have to digest the fibre, which in your case does not seem to be a good way. You could try to pipet the fibre with a small pipet up and down to strip the satellite cells of the fibre, but be careful not to damage the fibre. Have you checked for protocols in which the people isolate myonuclei and how they avoid contamination with satellite cells? Because not only satellite cells might be problem other cells for example fibroblast will be there as well.
I agree with Denise. In my case It has been about twenty years since I isolated satellite cells to grow but as I remember it involved digestion and d damage to the fiber (myocytes). Can you use fluorescent or immune fluorescence microscopic approach to both distinguishing the satellite cells from the myocytes and also for measuring drug uptake?
You may consider using myogenic stem cells and creating in culture myofibers (myotubes). I have been inducing myogenesis in C2C12 mouse myoblasts quite some time, it is easy.
Indem if you could an IF approach you can distinguish nuclei of Satellite cells from the rest. Satellite cells express PAX7 which is not the case for the myonuclei. Myonuclei in contrast should express for example myogenin. So in principle you could make nuclei from the whole tissue and for example FACS sort your myonuclei. I don't know if this is possible but would be an idea as well.
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