choosing the correct subculturing ratio - ovarian cancer cell lines - (Jun/08/2013 )

Hello,
I am quite desperately trying to find the right subculturing ratio for several ovca cell lines (A2780, HEY, CH1). Everybody in my lab seems to spli the cells in different ways - we do not have a standard protocol for them. The tricky thing is that they seem all happy but how can we know they really are...? We are trying to show a certain phenotype with knocking down a transporter gene but we only saw it in another (slower growing cell line) so far. However I am worried that the phenotype does not show up because we are mistreating our cells. We do colony formation assays and growth curves after siRNA transfection.
One of our fast growing cell lines is already at P28... Thats another problem we have. Can we still rely on such high passage numbers??

Thanks for any help!

If it's an immortal cell, then yes you can. We usually split 1:10 if we want to just subculture for later use. But we split with a bigger ratio if we have a lot of transfection to do in the week and need more cells. Most cells need 24 h to rest after seeding and before transfection.