4 layers instead of a monolayer in cell culture - (Feb/19/2003 )

Hello!

I am fairly new at cell culture, so I hope you will forgive me any stupid question!

I have a primary culture of epithelial cells from the bull epididymis seeded out in Nunc 0.4µm polycarbonate culture inserts. As I know from electron microscopy, the cells do not stop to divide, when they reach confluency, but rather grow in up to 4 layers over each other. That is bad, because I want to do some transport studies with a monolayer.
Culture medium is RPMI-1640 plus 1mM sodium pyruvate, 100nM Insuline, 200nM hydrocortisone, 200nM testosterone, 1 µM dihydrotestosterone and 10% FBS.
Cells growing over each other are definitely epithelial cells, for contamination with other cells is rare.
What can I do to get a monolayer?

Thanks a lot,
Y

try less FBS, like 2.5% cells will grow slower like that. as you may know with cell culture you have to keep ontop of them not become to confluent. from my experience it seems the best way to deal with cell culture is to never let them reach total confluency. plate them when they are 90%.

[COLOR=blue]When you resuspend your cells, do it with a yellow tip at the end of the pipet. Do 4-5 up and down carefully to break down aggregates. The best is to do that every time you have to plate your cells. Like the previous reply, I would tell you not to wait 100% confleuncy, and plate at 90%.