We seem to get a lot of proliferation in C2C!2 cells, even when they are treated with 2% horse serum. Though we see differentiation, there are still a lot of newly dividing cells in our cultures.
Dear Lakshman,
C2C12 are very tricky cell line, you must keep cells in culture at confluence less then 60%, otherwise you will select proliferating clone at the expenses of differentiatin one. Further in our lab we changed differentiation protcoll avoiding serum depletion and keeping C2C12 in growth medium, reaching colnfluence the cells form nicer and bigger myotube. You must play attention at growth confluence avoiding high confluence culture conditions.
Good Luck
https://genome.ucsc.edu/ENCODE/protocols/cell/mouse/C2C12_Wold_protocol.pdf
Hi Cassio,
Thanks for pointing to the paper, but, i had already gone though it. My question was not about the protocol, but rather about avoiding the newly formed cells during the differentiation process.
Dear Lakshman,
C2C12 are very tricky cell line, you must keep cells in culture at confluence less then 60%, otherwise you will select proliferating clone at the expenses of differentiatin one. Further in our lab we changed differentiation protcoll avoiding serum depletion and keeping C2C12 in growth medium, reaching colnfluence the cells form nicer and bigger myotube. You must play attention at growth confluence avoiding high confluence culture conditions.
Good Luck
Hi Lakshman,
I have good results with:confluence 80-90% switch to 2%HS DMEM, change media every 48h always at 37 degrees. If you grow them on coverslips (glass) you might not have as good results as plastic, but usually you have beautiful myotubes at 7 days with very few undifferentiated myoblasts. If you are growing cells on plastic, 10 days is the best. You may find different differentiation rates with different batches of horse serum.
Hope this help
S
I have found literature in support of both growth media, and 2% HS, as Sonia suggested. We have done our experiments with HS, and found decent differentiation. I was looking for a higher rate, though. Thanks for you suggestion, Cesare. Your point regarding the low confluency seems right. Will keep that in mind. Thanks for your suggestion too, Sonia. I think we will repeat this with 2% HS, as we tried earlier, and as you suggested. We have to see how it goes with 48 h media change. We kept a longer frequency for our experiments. Shall let you both know how it goes. Many Thanks to both of you.
Best Wishes.
Lakshman
If you feel that your HS is not achieving the results expected you have to change your HS to a different batch. I have a couple of colleagues that did so recently and all the problems were solved.
Good luck
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