more help: cell culture problem - (Apr/05/2005 )

I am culturing INS-1 cell now. Recently we got some problem.

1. After plating out 1 to 2 days, cell were found to become dark. When the cell reached high conflence, it became darker.

2. We also observed the cell contained some "vacuoles" in the cytoplasma. The cell membrane became soft and not smooth.

The medium we are using contains RPMI 1640 plus 10% FBS, 10 mM HEPES, 1mM Sodium Pyruvate, 2 mM L-glutamine, 50uM 2-mercaptoethanol and 1% P/S.

Did anybody here also meet this problem? Could you please tell me how to cure the cell?

Thanks!

hi
well i found a culture "protocol" which recipe differs little from yours regarding glucose :

RPMI 1640 with 11.1 mM D-glucose supplemented with 10% (v/v) fetal calf serum, antibiotics (10,000 units/ml penicillin and 10 mg/ml streptomycin), 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM {beta}-mecaptoethanol. (cline and al. jbc)