In my Lab is 2% horse serum, 0.1% insulin 5 days, changing the medium every 48h. I have seen several different protocols but didn't try until now
Hi Amaya,
Below is the link provides the information about C2C12 cell differentiation protocol.
https://www.encodeproject.org/documents/b42974fd-1490-4d51-bdcf-7db97a3e8fe0/@@download/attachment/C2C12_Wold_protocol.pdf
Hope this helps
Hi Amaya,
Below is the link provides the information about C2C12 cell differentiation protocol.
https://www.encodeproject.org/documents/b42974fd-1490-4d51-bdcf-7db97a3e8fe0/@@download/attachment/C2C12_Wold_protocol.pdf
Hope this helps
For differentiation, we switch to 5% horse serum, 1X penicillin-streptomycin in DMEM while C2C12 myoblasts is about 60-80% confluent. I prefer change fresh medium everyday for 5-7 days to get the best result (occasionally 2 days will be OK). The original C2C12 condition is also critical, if cells is stressed, it can be elongated but not fused to form myotubes.
Hi Amaya! I personally culture C2C12 cells in a Growth Medium (DMEM-GlutaMAX complemented with penicillin/streptomycin and 20% FBS) and I shift them in Differentiation Medium (DMEM-GlutaMAX complemented with penicillin/streptomycin and 2% FB) when they reach 60% confluence. They need to proliferate good to differentiate good, too. P.S. It is also a cheap protocol
In this paper we transfected the cells with a lenti-virus, but the results were still pretty good:
doi: 10.1155/2018/4814696
Let me know if it will works!
Alessio
Alessio Torcinaro hello, I am using nowadays the same but it takes more than 9 days to differentiate so is it normal?
and please I have another question, how we can measure the Differentiation Index (%), Fusion index (%), Nuclei per myotube (n), please I want the technical way to do that?
by way, I like the article that attachment it, it is so clear and easy to understand.
thanks so much
Hi Nour Mhd Nizar Al Zaeed ! Make sure that u make a fresh medium and that the components of the medium are not expired. How many cells do you plate per cm2? Which kind of plates are you using?
For the quantification you have to perform IF for MyHC (MF20) and counterstain with nuclei dye (e.g. DAPI). Differentiation index is the percentage of all MyHC+ positive cells, Fusion index is the percentage of nuclei per myotube, nuclei per myotube is the mean of nuclei into a myotube
I hope this helps
Alessio Torcinaro I usually used 2000cell /cm2 in T25 FLASK.
Thanks for all this advice, next week I will do it and I will make everything fresh.
Nour Mhd Nizar Al Zaeed your seeding density is very low. For 24 well and 12 well we seed this much opf density.For T 25 you can go to 20000 cells per cm2 as at conflency it will reach to 2.5 lakh cells.
Surabhi Dixit thanks for the reply I will take your recommended
thanks so much
Nour,
Do you check mycoplasma contamination routinelly?
It interfieres with normal C2C12 differentiation.
Best,
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