cell counting - (Feb/02/2011 )

Hi all,

New Bioforum member.

I've just begun cell culture work for the first time and have a question which is probably quite basic...

If i am growing cells in order to build up a stock for my experiments, should I count the cells each time i am freezing them down and aliquot them into the appropriate capacity for making up my next beaker? Or should I freeze down the contents from an entire beaker into one cryovial (T75 beaker:80-90% confluent).

Also, I am eager to make a stock of the low passae number I have, However, how can I do this if I only have a limited amount of cells to begin with and each time I reseed the cells the passage number increases! I'm fighting a losing battle.

Thanks in advance

Hi:
I am assuming the cells are adherant in a T75 flask. I freeze 5 cryovials from a nicely confluent T75 flask. I don't count them. On the label beside the name, date, passage, & initials I add T75 so I know when I thaw a vial I can put it into a T75.

Hi Again
You need to split the low passage flask to as many flasks as you can and successfully get the cells to grow. For instance: make 5 flasks from one confluent flask, let them get confluent then freeze 5 vials per flask. Gives you 25 vials or 20 vials (keep one flask growing).

lllueck on Wed Feb 2 11:31:04 2011 said:

You can do either, many people do count and freeze about 1x10⁶ cells per vial. This number should be enough to seed a t-25 pretty densely and a t-75 at about 10%, IIRC.

If you plan to use the entire contents of the beaker, then you can freeze them all together. However, it is always a good idea to save some cells from each passage in case there is a problem with the next passage. If you want to use only some of the cells, then aliquot them at the quantity you need for the seeding density of one flask or beaker.