Thread: Growing Sf9 insect cells in suspension culture.

Hi,

I have been growing Sf9 cells in suspension culture using TNM-FH medium with 10% FCS and glass Erlenmeyer flasks.
When I use 125 ml Erlenmeyer flask with 25 mL of culture medium, I observe the formation of a ring of cells on the wall of the flask at the interface between air and culture medium. However, the cell growth and viability sounds good.
When I use 250 ml Erlenmeyer flasks with 50 ml of culture medium, the ring of cells become more prominent and, sometimes, the cells in the ring start to die.
What should I do to prevent death of cells in the ring or the formation of the ring altogether?
I wash the glass Erlenmeyer flasks very scrupulously with Extran. After that, I rinse the flasks several times with water and Milli-Q water and keep the flasks with water overnight before baking, at 200 C for 4 h (to eliminate LPS), and autoclaving them.
Does it help to use pluronic F-68? Is there a problem with cells (that were a gift), should I buy new Sf9 cell from Invitrogen or somebody else?

Thanks,

G.Oliveira.