When culturing the L6 rat skeletal myoblasts, I usually change the culture medium (with 10% FBS and 10% horse serum) to differentiation medium (with 2% horse serum), and further culture the cells in differentiation medium for 2-4 weeks. But I never saw any beating myoblasts. Could someone give the solution to this problem, please?
I suggest embryonic stem cell differentiation to myobalsts first , then differentiation those myoblasta to contractile cells . May be the cell line were used could not adapted to differentiation medium. You can use other differentiation materials.
When culturing primary chick embryo myoblast they differentiate very well and undergo beating under brain extract addition to the medium. Try to use mouse brain extract (brain homogenization in PBS, 100000xg centrifugation and sterilization by filtering 0,45 micron) at 2% in differentiation medium. It might work.
Hi, Dr. Zani,
Thank you for your kind answer. I will try that in the future.
I have only cultured mouse C2C12 cells, but the same principle may apply. I found that the density at which you plate the C2C12 myoblasts plays an important role. Our lab plates the myoblasts so that they would be around 80-90% confluent on the day you want to switch to the differentiation media (we used 2% Horse Serum). Around day 4 and 5 we usually see spontaneous twitching and contraction. We never culture our cells more than 7 days though. I hope this helps.
Hi Man Mohan Shrestha,
Thanks a lot. It is very helpful.
I used L6 myoblasts of low passages (p3-p7). And I ensured cells did not reach over 80% confluence during subculture.
The medium you used (alpha MEM) is different from mine. In addition, you added NaHCO3 to the differentiation medium. I just searched some references and found NaHCO3 can enhance expression of myosin heavy chain. Is NaHCO3 usually used to to enhance differentiation in your lab?
Have you observed contracting myotubes by using your culture method?
Hi Man Mohan Shrestha,
Thanks a lot! Your information is of great help. I will try adding NaHCO3 in the differentiation medium.
Best wishes,
Shangwu
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