I would like to analyze separately protein from muscle mononucleated cells and from myofibers of the same muscle. My idea was to digest the muscle, as we do for mononucleated muscle cells extraction, and to keep the myofibers after the digestion with the collagenase. The question is: is it possible to use the waste of the extraction? Has anyone ever do it?
yes, is it possible and a little tricky. I did something like that in the past to analyze separately myofibers and satellite cells. I suggest to do a single fiber dissociation with collagenase, to obtain isolated individual myofiber explants. Then trypsinize them to separate satellite cells from them (you will need a lot of them for doing proteins, as freshly isolated quiescent satellite cells have a very small volume and compact cytoplasm with little protein content, in general, according to the protein of interest). In this way you will obtain mononucleated satellite cells and myifibers. However, If you are interested in analyzing also the other resident monucleated cells in the muscle tissue (endothelial cells, fibroblasts, mesenchymal stem cells, inflammatory cells, etc.) the situation is more complicated. I never tried to use the waste for extraction, but FACS sorting could be useful to isolate specific cell populations from the waste after collagenase, or at list to do an analysis to see what and how much and you still have there that you could use for protein analysis.
hope this helps,
Best,
Marco
- Badges
- Science topic