Hello everybody,
I'm trying to isolate and culture neonatal cardiomyocytes from mice using the protocol published on Jove and titled "Isolation and culture of Neonatal Mouse cardiomyocytes".
Application of this protocol has been successful the first two times, then I couldn't have any viable cell after the plating medium step on the 3rd and last day of the isolation procedure.
I checked the digestion step and I'm confident that nothing was wrong with that, so I can not easily find other possible causes. someone have any suggestions? I'm using media opened in July but they are not expired.
Thanks a lot
Michela
Dear Michela,
Would it be an issue to open a new bottle of media (at least leave this on the side and try using a new one, and then decide)?
CMs in primary cultures are very sensitive, as you know. I work with cell isolations and when an established protocol stops working, then I make new media, check the incubators (temperature, clean) and make new reagents and digestion solution, to star from the beginning.
A media bottle open from July could be contaminated, or some nutrients maybe not of the best quality, because it's been so long. Especially with the changes in temperature, from the different times you've used it.
If you want to be using a media bottle for many months, I would make aliquots and freeze them (make sure first if you can store them in freezer).
I hope this helps,
Sophia
Hi Sophia,
yes, your suggestion was actually what I also thought to do do. I changed all my media with new ones and the protocol worked again, anyway I still have a problem regarding the cells yield, that it's really low compared to how it should be following the protocol.
I played with the enzyme concentration as well as the time and the speed of the centrifugation step, but I still can not optimize that. Today I'll try to use a different kind of culture dishes.
Thanks for your reply!
Michela
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