C2C12 myotube contraction - (Aug/08/2014 )

Hi everybody,

I'm culturing and differentiating C2C12 myoblasts, together with a coworker of mine, and we're testing the effect of two media on myotube formation and differentiation. We've noticed that with the media we've been using to culture and differentiate the cells in until now, causes the cells to form myotubes very nicely (based on morphology). A month a go, we've had contact with a group in France who're also differentiating C2C12 cells, but they use medium that's different from ours in several aspects.

We want to enhance protein synthesis, basically. Where we see a protein enhancement of about 25% after stimulation with several components, they see an enhancement of up to 100% using the same components (in roughly the same protocol)! That's a very big difference. They sent us some photo's of their myotubes, and if we compare them with our own myotubes, we're prone to say ours look 'better' (in the sense that our cultures look as if they're further in the differentiation process than theirs, after the same amount of days).

We tested their media formulation against ours as a start of an attempt to recreate their results. So far, we see that our medium causes the cells to differentiate much faster. However, we also noticed that with their formulation, the cells show much more spontaneous contraction!

My question is: can you say that a culture containing myotubes that shows more contraction than another myotube containing culture is 'more healthy'? In other words, is contraction something you would want if you're performing experiments on myotubes? Can you judge the quality of the tubes based on how much contraction they show?

We are also working with C2C12 and our differentiation's 'quality' is kind of variable.

We normally check the differentiation degree by measuring myocyte markers by qPCR (MyoD, myogenin, and Myf5).

Regarding you specific question, I would say that the more twitching the healthier myotubes you have. Since you get such a nice differentiation...could you share the composition of you differentiation media?

Cheers

JM

Hi Adipo,

thank you very much for your reaction! We've also seen a lot of variation with regard to differentiation quality; between wells of the same plate and also between plates. We're having a hard time getting our results reproducible, because of this variation, although the variation is more in the amount and the shape of the tubes between wells. Sometimes the cells react very differently between experiments -  in one experiment they differentiate beautifully, and the next experiment they die off at the end of the differentiation period, even though the seeding procedure was the same. It can be very frustrating.

We've also been thinking about myocyte markers, so it's very helpful for us that you shared these markers. I've tried MyoD and myogenin myself, but I'm still optimizing the ICC protocol. Right now we're testing Troponin T as a myotube differentiation marker.

Regarding our differentiation medium: we used to do this in DMEM high glucose + GlutaMAX + pyruvate, with 2% heat-inactivated Horse serum and 1% pen/strep. This worked fairly well, however we then switched to DMEM high glucose + L-Glutamine instead of GlutaMAX, as we found in literature that the cells do not react well to GlutaMAX. The difference between these two media is huge - the cells differentiate much better in L-Glutamine.

So now we use DMEM 4,5 g/L glucose, L-Glutamine, pyruvate, 2% hiHS, 1% pen/strep.

Hi Susie Q,

Just wondering what the difference was between your media formulation and the other lab that had better spontaneous contraction?  I've heard that L-glutamine, heat inactivated horse serum, and no sodium pyruvate can make big differences.  So I'm wondering:

What amount of Horse serum did the other group use to differentiate thier cells and was it heat inactivated?  Also who was the supplier?

What DMEM did the other group use?  Did it have phenol red?  Did it have sodium pyruvate?

Did the other group use L-glutamine or Glutamax?

I'm trying to optimize spontaneous contractions and I'm running into a mess with people not reporting this stuff in their methods. 

Thanks sooooo much!!!