Trouble with differentiating L6 myoblasts - (Jun/12/2013 )

Hi all

I'm relatively new to cell culture and I'm having problems inducing my L6's to differentiate to myotubes. Here's the details:

I'm using 10mm dishes with 10mL alpha-MEM, 2% fetal calf serum and 1% penn/strep.
Differentiation media added when cells were roughly 80% confluent. Cells are passage 14.
I could just be expecting things to happen too fast, but 2 days later there appears no change in the cells, and they have contuinued to proliferate (ie most of my plates are bordering on 100% confluent). Now seeing some cell debris on the surface which I'm guessing is from cells dying. No detected contamination, media pH is fine and no turbidity.

Is this normal or should I be seeing some movement towards alignment? I'm going to change the media today but was wondering if it is worth passaging the cells to give them some more space. I'm aware it can take ~10 days to diff but after 2 the only difference is that there seems to be more blasts!

Sorry if this is newbie stuff - any help suggestions much appreciated

I work with C2C12 cells. Read this paper, it may be helpful.

http://www.daba.lv/g...ksts_2000.pdf


It sounds like you are doing everything correctly. I would wait a couple more days. I would grow five plates of myoblast cells and harvest RNA at undifferentiated, D2, D4, D6, and D8. Before you harvest the RNA, take a picture of each plate in a couple of different areas. Use the RNA to run a RT-PCR on common differentiated and undifferentiated genes (MyoG, MyoD, MCK, etc.). This will give you a template to reference in the future and you will know to what extend your cells are differentiated from your RT-PCR data.

My only concern is that your cell passage is relatively high. When cells are passaged to high, they can exhibit irregular patterns of growth. We usually only use cells to passage 10ish. The lower passage the better. If you have lower passage cells (1-3), grow a large plate out and freeze 10-15 vials for future use.

Good Luck

Edit:
The above link is dead.

http://www.daba.lv/grozs/Mikrobiologijas/Biol%20Akt%20Probl/Biol%20Aktual%20Probl_Pavasaris%202012/Journal%20club/09_Barba%20Ieva%20Tatjana_Elina_Dace/Elina%20Buraka_raksts_2000.pdf

Paper name:
Differentiation of Myoblasts in
Serum-Free Media: Effects of Modified
Media Are Cell Line-Specific

Good reference website:
ATCC.org

Thanks jerryshelley. I had seen that paper but will give it a better look, at cursory glance it seemed to me that it suggested serum-free media worked for C2C12s but not for L6s (skim-read!).
Now at 6 days and they are starting to fuse so I think I was just impatient! I had my cells donated from another lab at P9, and they said they usually have no problems up to passage 20-25. I did exactly as you suggested and have frozen about 30 vials of P10s, but I'll look at getting some fresh cells.

Good tip for running PCR, pity I didn't see this earlies (so already at D6), but I'll look at doing this soon.

Thanks for your help