Thank you in advance for your help. My myotubes are plated in 12 well plates coated in Matrigel in 10% FBS DMEM and allowed to reach confluency before being switched out to 1% FBS DMEM (day 1 of differentiation). Media is changed every other day and the myotubes look great around day 5. They cover the entire plate and are straight/long.
Around day 6-7 they begin to start acting weird and I don't know why. The myotubes become branched and fuse together in different directions, and ultimately they die. The way they fuse is almost a mesh-like pattern.
Has anyone else had this problem? I've checked the incubator for correct temp and CO2. It's not contamination, either. Thanks!
Dear Jj,
you can improve the C2C12 attachment by pre-coating your dish/wells with a suspension of Matrigel diluted 1:80 in DMEM.
Regards
Hi Ji,
C2C12 are quite tricky, for long term myotube formation you must uise DMEM Gibco, I had the same probably changing medium brand. Moreover, you can try to acoid serumn depletion, C2C12 differentiate in large myotube reaching over confluence.
Good luck
Hi Ji,
I've had the best results when I initiate differentiation at about 80% confluence and by using 2% horse serum instead of 1% FBS. However, even then I found that the cells remain stable only for about 2 weeks after seeding.
Best of luck
As already told you I would differentiate myotube in DMEM Gibco and Falcon Plasticware, and then try to let the cells differentiate in complete medium rather then deplete serum.
Good luck
Dear Jj,
you can improve the C2C12 attachment by pre-coating your dish/wells with a suspension of Matrigel diluted 1:80 in DMEM.
Regards
Hey Jj,
I don't coat my dishes at all. At what glucose concentration do you differentiate them? I found that I can get a few more days out of them by reducing the glucose from 4.5g/L to about 2.5-3g/L. However, even then, in my experience the C2C12s aren't really suited for such long-term studies. Why are you trying to differentiate them for so long?
Hi Stefan,
I'm differentiating them at 4.5 g/L. I have some low glucose DMEM so I'll give that a shot. We are going to be stimulating them with LPS, but I'll link a study that showed that it can inhibit the differentiation of the myotubes. I want to make sure I can get them terminally differentiated before hitting them with LPS
Article Lipopolysaccharide inhibits myogenic differentiation of C2C1...
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating