# seeding density - questions! (Dec/04/2008 )

I am working with a cell line that I got from ATCC. The documents that came with this cell line indicate that when subculturing an inoculum of 6 X 10(3) to 1 X 10(4) viable cells/sq. cm is recommended and cultures should be maintained at a cell concentration between 2 X 10(5) and 5 X 10(5) cells/sq. cm.

What exactly does this mean?

Check this post for the area of the wells.

http://www.protocol-online.org/forums/inde...ost&p=64847

"when subculturing an inoculum of 6 X 10(3) to 1 X 10(4) viable cells/sq. cm"

means you need to put 6 x10^3 to 1x10^4 cells per cm^2

You may need to calculate how many cells to seed into each flask/ well.

"should be maintained at a cell concentration between 2 X 10(5) and 5 X 10(5) cells/sq. cm."

means if the cell density reaches about 5x10^5 cells per cm^2, you may need to subculture it.

Normally, people subculture the cell line when it reaches 80% confluent.

Hope this may help.

Thanks Minnie Mouse, but the recommendations from ATCC seem pretty dilute after I calculated it out. About 150,000-250,000 cells per t-25 flask. Does that sound right??

yes.