I am growing myotubes in 96 well plate for treatment with free fatty acid. However, they come off after washing or even treatment with some dye for 15-20 minutes in a buffer. This is with myotubes without treatment as well. It was not happening earlier. I observed that cell crowding at the edge of the well might be the issue, so instead of 4th day I started using 3rd day old myotubes, but still there is no change in the behavior. I am wondering how I can control this? Do you have any help?
Are you treating the surface of your plates with any coating? C2C12 usually don't need anything on cell culture plates besides what it already comes with out of the package, but since they are mature myotubes they may be using up what they can stick to on the plate very quickly. It's a shot in the dark, but maybe try coating your wells with 0.1% gelatin, or a similar substance so the myotubes have a bit more to attach to. What do muscle fibers usually attach to in vivo? A coating that has similar factor components to the in vivo environment may help as well.
Good luck!
Hmmm... That's a good suggestion.. But you know there was no problem earlier.. Suddenly started.. May be try this one.. Thanks Jessica !
Hi Madhurima,
Actually I use a new vial for a new experiment. And as you said they adhere very well to the plastic dish. I don't see what suddenly has gone wrong. But I will try coating the plate. Also, I would like to know.. if I left the cells to form myotubes without changing differentiation medium everyday, will that form stronger myotubes ? And how many cells you seed in 96 well plate? I use cell density of 10000/ well.
Thanks and waiting for your response !
Rashmi
Myotubes usually don't need any change in medium. C2C12 are clonal originally, but they change gradually their phenotype until they reach a tumorigenic state. this is not your case as tumorigenic C2C12 don't differentiated anymore. Anyway, when I use C2C12, I usually reclone them : plate one cell per well, amplify, test for differentiation and attachment, and further amplify and freeze down clones which have a good behavior. A bit long, but a security to make experiments with cells in a good shape and a proper differentiation for a whole set of experiments.
HI Madhurima,
OK, then I will try coating the plate. 1000 cells/ well isn't too low a density to start with ? Just asking..
Thanks,
Rashmi
Hi again !
I tried doing cell number variation, 1000 and 5000, but again the same problem :( I didn't coat the plates, just wanted to see if cell number will solve the issue. Also, intuitively I feel coating is not an issue, as earlier cells were growing fine. What to do ??? Just would like to know, do you coat your plates ?
Hi Rashmi,
You have tried many things Better to barrow some cells from other lab may be your stock has some problem. In past we also faced the same problem than we purchased new cell lines than they worked fine. Coated plates are not necessary for the differentiation of C2C12 cells .Are you treating cells with 2% FBS for differentiation. Better to seed 2X105 cells and treat cells next day.
Hi,
I think I have got the solution, cells are doing OK now. I tried gradually reducing % FBS in the differentiating medium to 1% and 0.5%. And they are doing fine. Problem was I guess even 2 % FBS was inducing proliferation.
Thank you all for your suggestions !
Hi Madhurima,
You suggested that if I want to see beating myotubes and measure their contraction, change media up to 6-8 weeks. What is the composition of differentiation medium (DM) do you use, please? How much volume of DM do you add to a 96-well? Do you change the medium every day?
Thank you very much.
Shangwu
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