Hi everyone,
We are currently doing C2C12 cell differentiation and unfortunately, even after 4 days in the differentiation medium, the cells elongated but we do not see cell fusion at all. We stained cells with MyHC and all the cells are MyHC positive and single nucleated.
We used just a simple protocol for differentiation with DMEM, 2% heat-inactivated horse serum, 1% PenStrep, and the medium is refreshed every other day. The C2C12 was from the other lab and we don't know which passage they were in.
Could anyone suggest the possibilities that can happen to affect C2C12 cell fusion to form myotubes?
Hello Mave Tran
Are you using DMEM with high glucose? Add 1% L-glutamine as well.
C2C12 cells need to be at a low passage number for avoiding reduced differentiation rate. You can try supplementing the differentiation media with insulin/transferrin/selenium. Change the differentiation media every 48 hrs.
Hope this will help!
Best.
Hello Malcolm,
Thank you for the kind reply. Yes, we are using high Glucose DMEM. May I ask what is the reason for using 1% L-glutamine or insulin/transferrin/selenium? I found people used DMEM with 2% HIHS and it worked well.
Yes. I found a lot of recommendations related to cell passages and low-density maintenance. I think we let them be confluent several times before using so that might be the reason why. We are currently trying again with the new stock of cells and hope they will work fine.
It can take longer than 4 days. In my experience it never takes less than 5 or 6 days, and usually 7-10 days is the sweet spot for half-decent myotubes.
Growing myotubes isn't easy: once they've fused and aligned nicely they start getting contractile pretty fast, and once contractile they're very likely to pull themselves off the plate surface. The window between "not ready" and "too differentiated to be useful" can be quite small.
What are you growing them on? Collagen? Matrigel?
And what antibody are you using to label MyHc? Myogenic cells shouldn't usually start making contractile proteins until they're fused and quite far along the differentiation pathway, so getting robust expression in mononuclear cells seems...unusual.
Dear Trinh,
Differentiation of C2C12 to form multinucleated myotubes can take more than 4 days. But usually, if you use a low passage cell (P1-P14) it starts fusion from 3 to 4 days. As a differentiation media you can use 0.1% insulin (1microM) in them. (DMEM+2% Horse serum+ 0.1% insulin solution+ 1% anti anti or pen strep). Try using this media composition. Insulin triggers cell differentiation. But the low passage is the highest priority.
Hi John,
Hi Madhura,
Many thanks for the answers.
I totally agree about the early passage cell benefit in differentiation. I did try to differentiate C2C12 at early passage and got quite a nice fusion only 3 days after inducing. They fused and aligned nicely, but It was years ago in my former lab.
Right now, my colleague isn't sure about the passage of the provided cell stock. We are trying to differentiate them for a longer time and we noticed that there are a lot of cell deaths as well. We grow them on just a normal 24 well plate without coating.
Yes, we stained the cells with MyHC. And John, your point is also what I am worrying. That's why I think the cells somehow couldn't fuse. But we definitely should try for a longer time and see what happens. Will try to add Insulin as Madhura and Malcolm suggested as well.
Thank you.
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