Lately, I've been having a lot of difficulty differentiating L6 myoblasts and I'm unsure what I'm doing wrong.
After adding the cells to 6 well plates (200,000 cells per well) and allowing them to grow for 48 hours in growth medium (GM: AMEM supplemented with FBS and antibiotic-antimycotic agents to final concentrations of 10 and 1%, respectively), I wash with PBS and shift the cells to differentiation medium (DM: AMEM as above except that 2% HS replaced 10% FBS). Within 48 hours of differentiation, the majority of the cells die and the remaining cells differentiate very poorly such that no myotubes form by day 5 of differentiation.
The only thing that I can think of is that the wells are too confluent by the time that I transfer the cells to DM. Attached I've added a picture of a well just prior to transferring it to DM - you can see that it is nearly 100% confluent.
Any help would be greatly appreciated!
Mehras,
We had a similar problem trying to differentiate c2c12 myoblasts. It turned out to be an adhesion problem. During fusion, the blasts would come off the plate. So, we started gelatin coating our plates. We haven't had problems since.
Gelatin coating culture plates is very easy, inexpensive, and cane be done days/weeks ahead of time. It might be worth a try.
Good luck.
Mehras,
I think that you can replace AMEM medium by DMEM with 4.5g/L of glucose. The density seen in your image is the better density to induce différentiation. Try the same density and high glucose DMEM with 2% hs, in C2C12 cells after 4 days of differentiation a 40 to 60% index fusion is usually observed.
We used to have differentiation with 2% FBS, but, several months ago, we started to have the same problem. We have tried with 2% HS and had just a little bit of fusion. How many passages have the cells? Maybe you have to get a vial with low passages. Another option is low down FBS to 1% since the quantitive of proteins is usually different between serums.
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