when to start counting passage number for primary cells? - Bovine aorta endothelial cells, for example (Aug/06/2006 )

Hi,

I am doing some primary cell works using bovine aorta endothelial cells. I harvested some cells from aorta, cultured them for about a week and picked colonies up using cloning rings. Now I put these in 24 welll plates to grow them up, and paln to expand them through 6 well plates, then 100 mm dishes. My question is when do you start to count the passage number? Is there a consensus for that practice, or just arbitural one? Thanks.

I have the feeling it's arbitural.
I start myself counting one passage when I split the first time.
It means that in the first flask I have a primoculture, and then I trypsinize and count one passage for this new flask.
I think some people already start to count one at the first flask.

Dear Genehunter-1,

When isolating the BAEC's from tissue using collagenase, spinning them down and plating them out, they are at this stage Primary's. When they grow and require trypsinisation for the first time, they are then at Passage 1. It is important to keep an accurate record as the cells will definetely change over time, they even change pharmacologically from Primary--P1.
We had to use P1 cells only for our experiments, so I was constantly having to go to the abattoir to obtain fresh tissue.
Also another thing to be wary of is the use of Antibiotics/Antifungals....these potentially can have massive effects on the BAEC's.