Hi everyone,
I am trying to optimize C2C12 differentiation protocol for studying muscle disorders. I am using following conditions for culture
1- Proliferation medium is 10% FBS, 1% PS in DMEM High glucose and cells were cultured in 100mm plates. At 50% confluency, cells were trypsinized to 6-well culture plates.
2-Upon 90% confluecncy, differentiation meduim was used which is 2% HS, 1% PS in DMEM high glucose and differentiation was continued for 6 days and media was changed after every 24 hours
Following are my questions
1- I was able to get only 70% myofibers formation and remaining 30% were simply the c2c12 cells which were not differentiated to myotubes or myofibers. So, can I treat my cells here at this differentiation to get optimum atrophy markers like Murf 1 and Atrogin 1? or there should be 100% myotubes formation?
2- I am trying to use 10uM Dexa for muscle atrophy establishment and I treated my cells with Dexa in 10% FBS in DMEM high glucose and i obtained non-significant increase of Atrogin 1 in Dexa treatment group which is worrisome for me. So, should I treat my cells with Dexa in serum free media to get significant increase of atrogin 1?
i am also attaching a picture of my culture. please guide me that cells at this stage are okay to use or not in terms of myofiber formation
Regards
Arif
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