Hi,

I had the same issue, tried different plates coated with poly lysine, fibronectin, also ibidi plates but nothing worked for me. After tryiing out different dishes, I finally found the issue. 

I seeded c2c12 cells on a glass bottom dish, allowed them to reach confluency, once they became confluent i washed them twise with PBS and changed to differentiation media containing 1 micromolar insulin. I think insulin here is the catch, that some how made the myotubes to stick to the glass. I changed media once every 2 days, after 7-10 day i got nice myotubes. 

You will also notice along the edges of the glass plates myotubes start to come off, but ignore them. you will have enough cells in the middle to take pics. Handle them gently from differentiation state till you fix and stain them.

I tried the same protocol on ibidi dish. Got stunning pics.

Differentiation media (2% horse serum and 1 um insulin)

I can send you pics if you want

Thanks for your answer!

Indeed I thought about that as well but the thing is IBIDI chambers are indeed very expensive and the amout of antibody to do IF will be increased very much. Moreover I can image the cells only once. Thus I really want to stick to my coverslips. 

Concirning the spontanous differentiation. I look very specific into the first days of fusion and I need a more or less syncronzied differentiation, thus just letting them in the medium is no option for me. Anyway I do not have problems with the cells dying. They differentiate very nice they just rip of the collagen from the coverslip during fusion and then detach.

Matrigel is another (albeit expensive) option. I am assuming you're using poly-lysine coated coverslips before you add the collagen/laminin? It helps a bit.

In my experience, though, there aren't many ways round this: myotubes simply don't like glass very much. I don't think it's a collagen depletion so much as a constant force exertion on loosely attached growth surface: they literally pull themselves (and the collagen matrix) off the glass.

You could try seeding and differentiating them at a lower cell density, so the tubes they form are more isolated and less "one cohesive sheet", or (depending hugely on exactly what level of detail you're hoping to glean with your immuno) simply do the whole thing in plastic 12-well plates and use an inverted fluorescence microscope. Yes, plastic is not ideal, and yes, it autofluoresces somewhat, but it does still generate usable data.

You fix your myotubes (PFA, not cold acetone, obviously, beause...plastic), permeabilise, etc. Basically do everything you would with coverslips, but just on plastic, in wells. Then stick a coverslip on top (or even just image while covered in a small volume of PBS).

You could also try chamberslides, which come in glass or plastic varieties. They cost a fortune, but I know myotubes do form on them, and you can get a lot of different samples on a single slide. (edit: I note Vasily already made this suggestion. Whoops)

Hi Denise, have you resolved the issue of  detachment of differentiating c2c12 from culture surface (for imaging) ??  I too am facing the same problem. 

Hi,

I am also trying (just a few times) to differentiate C2C12 cells on PDMS substrates coated with fibronectin or laminin but I observe a detachment of the myoblasts, starting at the borders of the monolayer, before reaching a good differentiation. I think that it is the same problem that you had. Did you find a solution? Was polylysine a good solution? Thanks!

I found no solution I switched now to normal culture dishes andimage with a LD Objective.

Hi,

I had the same issue, tried different plates coated with poly lysine, fibronectin, also ibidi plates but nothing worked for me. After tryiing out different dishes, I finally found the issue. 

I seeded c2c12 cells on a glass bottom dish, allowed them to reach confluency, once they became confluent i washed them twise with PBS and changed to differentiation media containing 1 micromolar insulin. I think insulin here is the catch, that some how made the myotubes to stick to the glass. I changed media once every 2 days, after 7-10 day i got nice myotubes. 

You will also notice along the edges of the glass plates myotubes start to come off, but ignore them. you will have enough cells in the middle to take pics. Handle them gently from differentiation state till you fix and stain them.

I tried the same protocol on ibidi dish. Got stunning pics.

Differentiation media (2% horse serum and 1 um insulin)

I can send you pics if you want