Recently,I work with c2c12 cell.I found they grow very well in growth medium ( high glucose DMEM+10%FCS),but when diffentiation medium(2%horse serum) is added,the next day,lots of them died,even at thirth day.what puzzled me best is that after being treated with 0.2%BSA ,myotube looks better than in 2%horse serum.Anyone can explain it?Thank you very much!

Have you tried a different lot#/batch of serum?

Thank you samm.
I use horse serum without heat inactivating,they grow very well before,and often I could see them constracting .Our DMEM contains 25mM glucose ,glutamine, HEPES,NahCO3,penicillin and streptomycin.Horse serum came from gibco.I use 2%horse serum as differentiation medium.

If your 0.5%BSA (+2% horse serum) looks better than your 2% horse serum alone, it leads me to suspect the serum.
As I mentioned earlier, you can try warming the horse serum to 56dC for 30 mins (no warmer, and no longer) in case its a C'mediated effect.
Also, since BSA actually makes your cells look better, there could be a lipid/fatty acid imbalance.
You could try asking GIbco/Invitrogen for a sample of a different batch of horse serum - they are usually quite happy to oblige.
All the best

I will try your suggestion,thanks again!