Hi!

Firstly, you're using the right media, so thats good! DMEM (High Glucose), 2% HS and 1% P/S.

I have had a lot of success differentiating C2C12s. Normally, once I get cells to 90-100% confluence I change to differentiation media. But once cells are in DM, you DO NOT change media daily (I thinks this is what you're eluding to in your question?). Once in DM, leave cells in DM and don't media change. Cells will ultimately differentiate due to low serum conditions so if you keep replenishing media, they wont differentiate effectively.

Another couple of trouble shooting steps could include:

-Plate cells on laminin

-seed lower passage cells

-check plastic is suitable for adherence

-potentially lower serum even more (incramentally - 0.25% each time?)

Sorry if I misunderstood your original question. I hope I have been of some help!

We tend to initiate differentiation at a slightly lower confluence (about 90-95%%) and we too, switch out the differentiation medium every 48hrs with warm medium. Myotubes, as you said, are usually fully formed in 4-6 days. When the confluence gets too high, you can get early differentiation as the myoblasts start to fuse together and the media conditions aren't optimal at that point, leading to smaller, less differentiated fibers (so we don't usually go all the way to 100%)... and there is just less room for the fibers to grow and extend out.

I didn't see it in your protocol, but we also found that many of our cells do not adhere well to normal plastic cell culture plates. We coat our plates/wells with gelatin (0.1% gelatin (sigma G9391): 0.5 g gelatin in 500 ml ultra-pure H20, autoclaved at 121C, 15psi for 45 min, store at 4C) To coat our plates, we warm the gelatin solution to 37C, and add just enough to each well/plate to cover the bottom of the plate. We incubate at room temperature (in biosafety cabinet) for 1 hour and aspirate excess gelatin. After the plates completely dry, they can be stored for up to one month at 4C (though they should be warmed to room temperature prior to adding the myoblasts that will be differentiated).

I hope this helps with your differentiation endeavors, good luck!

Rightly answered above but in general cell lines do not need coating the culture well. Confluence is important as that would determine fusion of cells (distance between each cell) which eventually determines the quality and quantity of differentiation into myotubes. As mentioned above, if you start earlier (70-80% confluence) cells would start forming myotubes around day 3 of differentiation but myotubes are evident on day 1 or 2 when differentiation mediated is added at 90 to 95 % confluence.